Identifying the biosynthetic origins of nybomycin, a reverse antibiotic

The emergence of multi-drug antibiotic resistance in many disease-causing bacteria has raised the prospect of a devastating return to the pre-antibiotic era. It is imperative that new antibiotics are developed to counteract this problem. More than 50% of clinically used antibacterial agents are of natural product (NP) origins, with the majority produced by harmless soil bacteria called Streptomyces. We propose to study the Streptomyces natural product nybomycin.

Nybomycin was first discovered in 1955 but does not dissolve in water and was not used for drug development. Very little further research was performed on until 2012 when, it was "rediscovered". Surprisingly, it was found to be active against a strain of Staphylococcus aureus with multiple resistances to antibiotics (MRSA) but not against an antibiotic sensitive Staphylococcus aureus (wild-type) strain. Further study showed that nybomycin only killed bacteria that were resistant to a class of antibiotics called quinolone antibiotics. Quinolones kill bacteria by preventing their DNA from unwinding and duplicating. This unwinding is controlled by special enzymes called topoisomerases (aka. gyrase) , and nybomycin was shown to be inactive against strains carrying normal topoisomerase genes but active against those with mutated genes resistant to quinolones.

All bacteria will eventually develop resistance to whatever antibiotic they are exposed to. However, when strains of MRSA were grown in the presence of low levels of nybomycin, in order to select for nybomycin resistance, they became susceptible once again to quinolones by reversion of the relevant mutation. Due to this phenomenon nybomycin was termed a 'reverse antibiotic'. Reverse antibiotics offer the opportunity to stop the cycle of the accumulation of endless resistances, where strains of bacteria pick up more and more antibiotic resistances each time they are treated with a new antibiotic. In the case of nybomycin, it is a two-step process: bacteria resistant to quinolones are treated with nybomycin and any that develop a resistance to nybomycin are killed by quinolones.

Since nybomycin is not soluble in water, it is not amenable, in its current form, to being used as an antibiotic to treat infections in humans. The aims of this research are:

1. To identify the genes responsible for the production of nybomycin;
2. To discover how nybomycin is made in the bacterial cell;
3. To understand how nybomycin kills MRSA and other quinolone resistant bacteria.

To accomplish these aims we will use the genome sequence of the bacteria which makes nybomycin and identify the genes responsible for making it. We will then delete these 'biosynthetic' and 'regulatory' genes from the bacterial genome one at a time. In some cases we will insert extra copies of the genes. After each experiment the production of nybomycin or new, related compounds, will be analysed to see which changes increase or decrease production of nybomycin or if any new compounds are produced. This will allow us to determine the sequence of events leading to nybomycin production and the function of the gene products (enzymes) involved in this process. Finally, we will study nybomycins mode of action by testing its ability to inhibit the action of various gyrase enzymes as well as the ability to kill a range of different bacteria.

As part of the research we anticipate isolating nybomycin like molecules that may be starting points or the inspiration for developing an antibiotic active against MRSA.

The Streptomyces natural product nybomycin was first reported in 1955 but was found to have variable antibacterial potency, be extremely insoluble in water and did not find clinical utility. It was rediscovered in 2012 during a screen for substances active against multidrug-resistant Staphylococcus aureus M50 which identified an actinomycete fermentation extract with a curious activity profile: nybomycin was inactive against strains carrying intact gyrase genes (quinolone sensitive) and active against those with mutated genes (quinolone resistant).

Unexpectedly, rather than select for further additional mutations to generate dual resistance, quinolone resistant strains treated with nybomycin reverted to quinolone sensitivity by reversion of the relevant mutation. Due to these phenomena nybomycin was termed a 'reverse antibiotic'. Preliminary stable isotope feeding data from the 1970s suggest that nybomycin biosynthesis does not conform to any general, known biosynthetic pathway and likely comprises novel chemical and biochemical mechanisms.

Taken together these observations make nybomycin an excellent topic for an interdisciplinary project comprising a range of microbiological, genetic and chemical methods.

Research plan:

1. Identify the nybomycin biosynthetic gene cluster using transcriptional analysis and/or transposon mutagenesis;
2. Determine the nybomycin biosynthetic pathway by a combination of mutational analysis, heterologous expression and biochemical characterization of pathway enzymes;
3. Investigate the mode of action using DNA supercoiling and cleavage assays for quinolone sensitive and resistant gyrase enzymes from multiple organisms.

Nybomycin has been shown to be active against MRSA, VRE and Tuberculosis and has the potential to be active against other Gram-positive quinolone resistant pathogens. Our research aims to:

1) Identify the nybomycin biosynthetic gene cluster
2) Delineate the biosynthetic pathway and explore its manipulation; this may lead to the production of new, structurally related molecules and provide information to enable increased production titres
3) Increase our understanding of the way nybomycin acts on DNA gyrase

We believe that our research has the potential for far-reaching health, economic, societal and academic impacts - not just through the results of our primary research, but through the associated AMR related outreach and engagement programme.

The development of a nybomycin based antibiotic would be of value to the pharmaceutical industry. Streptomycetes account for ~80% of commercially important antibiotics used in human medicine, and are also a rich source of other types of bioactive molecules such as anticancer agents and immunosuppressants, currently accounting for ~$40 billion of revenue annually in the pharmaceutical industry worldwide.

According to the O'Neill report on AMR (May 2016), 700,000 people a year die of AMR infections every year. Apart from this tragic human cost, the report estimates that if action is not taken, by 2050 the cost in terms of lost global production will be 100 trillion USD. The current financial burden of MRSA alone in the UK is considerable. In 2008, Marsh Risk Consulting estimated that it costs the NHS £45 million annually to keep patients in hospital to treat them for MRSA. A more effective treatment could reduce this cost substantially.

In addition, a dual nybomycin/quinolone treatment approach to combat MRSA infections would mean that "last line of defence" drugs, which are increasingly being used more frequently to treat MRSA, could once again be retained for emergency cases. This would prolong their efficacy and help to delay the post-antibiotic apocalypse predicted by many. In addition, since resistance to nybomycin results in sensitivity to quinolone antibiotics, there is significantly lower risk of creating new "superbugs" with multiple resistances.

Furthermore, preliminary data shows that nybomycin is also effective against the causative agents of tuberculosis. The societal, health and economic impact of a new treatment for tuberculosis would be enormous. The WHO estimates there were ~480,000 new cases of multi-drug resistant tuberculosis in 2014 and there are an estimated 1.5 million deaths by TB each year.

Apart from the obvious economic, health and societal impacts associated with the development of a successful new antibiotic we believe that the AMR specific outreach and public engagement associated with our project will lead to an increased awareness and understanding of the problems associated with misuse and over use of antibiotics. This is turn will lead to a shift in behaviour and attitudes towards more responsible and informed use of antibiotics.

We have a strong track record in engagement with primary and secondary school children through science festivals, JIC science camps and through the SAW trust workshops and we plan to continue this throughout the project. The O'Neill report on AMR (May 2016) specifically recommends a "...global public awareness campaign to educate all of us about the problem of drug resistance and in particular children and teenagers". Apart from the impact of raising awareness in the next generation of antibiotic consumers, we believe we will also inspire the next generation of scientists through our engagement and outreach.