When Matthew (Matt) Dunne arrived at EMBL Hamburg from the University of Birmingham four years ago to start his PhD he wasn’t planning on working on bacteriophage enzymes. “The work on bacteriophage endolysins actually started as a side project to help with developing my lab skills at the start of the predoctoral studies” he smiles. As often is the way in science, events took a different turn, and the little side project evolved to shape the rest of his PhD and his future career. “I want to stay in the phage field” he says. Matt looks relaxed as he sits opposite me. He should be – he has just successfully defended his PhD, his work on bacteriophage endolysins is published today in the open access journal PLoS Pathogens, and he has even managed a very decent 36th place in the recent Hamburg Triathlon.
Using structural biology methods Matt and his co-authors have been able to show how the bacteriophage enzymes that destroy the bacterial cell wall – known as endolysins – are activated in Clostridia species. A crucial part of the bacteriophage life cycle that was not well understood until now. Matt worked on bacteriophage endolysins specific to two Clostridia bacteria, and has been able to show that the enzymes switch from a tense, elongated shape, with two endolysins joined together, to a relaxed state where the two endolysins lie side-by-side. The switch from one conformation to the other seems to release the active enzyme, which then begins to degrade the cell wall. Surprisingly Matt observed the same mechanism in both of the two Clostridia bacteria species he was studying, and so he and his colleagues believe this could be a more widespread tactic among bacteriophage endolysins. If so, this would be a real breakthrough in bacteriophage research and enable the development of bacteriophage technologies in a whole range of areas such as health, agriculture and food industries.
“It’s been fun” Matt says looking back. “It has definitely made me want to stay in science for the time-being. I really like the freedom you get with science. It’s like getting paid to do a hobby – a stressful hobby at times though!” he grins. He now looks forward to spending the next few months finishing off this fascinating story before moving on to pastures green. “There will be a few more twists and turns, I am sure” he adds.
The problem of antibiotic resistance
In the light of increased bacterial resistance to antibiotics, bacteriophage research is now getting more attention. Antibiotic resistance is a problem. A big problem. Not in some far flung land but here on our doorstep, and not for just the old and frail, but everyone – you, me, our children. In a recent press release, Dr Keiji Fukuda, Assistant Director-General for Health Security at the World Health Organisation (WHO) stated that “without urgent, coordinated action by many stakeholders, the world is headed for a post-antibiotic era, in which common infections and minor injuries which have been treatable for decades can once again kill.” This is not a surprise or even new –Alexander Fleming who discovered the antibiotic penicillin in the 1920’s was fully aware of the problems of antibiotic resistance. “It is not difficult to make microbes resistant to penicillin in the laboratory by exposing them to concentrations not sufficient to kill them, and the same thing has occasionally happened in the body” he explains in a lecture held on the occasion of his Nobel Prize in 1945. “…there is the danger that the ignorant man may easily underdose himself and by exposing his microbes to non-lethal quantities of the drug make them resistant…. Moral: If you use penicillin, use enough.”
Wise words and ones which we obviously should have heeded better. Now increasing bacterial resistance to antibiotics threatens to change the very world and lifestyle that antibiotics was able to create. What would life be without antibiotics? Major surgery, cancer treatment, tooth ache, seasonal bugs, a walk in the woods, a tattoo even – all suddenly carry a greater risk, and become potentially lethal. This doomsday message is finally slowly seeping through to all levels of our society – in Britain the “Longitude Prize” was recently launched where public voters chose antibiotic resistance as “one of the greatest issues of our time”. Ten million pounds will be distributed to successful project proposals this Autumn. International taskforces have been established to monitor and fight the problem, Health organisations are doing their best to make us aware of the consequences of antibiotic over- and illuse, but we still need a solution and we need it soon.
Bacteriophages – an alternative to antibiotics?
While some research looks at prolonging the lifespan of currently still effective antibiotics, other alternative avenues are also being explored. Bacteriophages, viruses which attack and destroy bacteria, have long been thought of as a possible alternative to antibiotics. Discovered almost exactly a century ago, bacteriophages were manufactured in Europe and America by the 1930s but were then forgotten about in the western world as antibiotics were became popular in the 1940s. Now, in the light of increasing antibiotic resistance, bacteriophage research is going through somewhat of a renaissance. Unlike traditional antibiotics which also kill friendly bacteria, bacteriophages target specific species, or even strains. What has never really been understood, however, is just how the bacteriophage destroys the bacteria cell, in particular how the bacteriophage “endolysins” – enzymes that break down, or “lyse” the cell wall – are triggered to carry out the destruction. This has been the missing part of the puzzle. Know this, and we have the knowledge to engineer effective and specific bacteriophage treatments to fight antibiotic resistant bacteria.
Four years ago, Matt’s supervisor, EMBL Group Leader Rob Meijers, had already established connections with collaborators at the Institute of Food Research in Norwich, UK and the first structures of bacteriophage endolysins specific to Clostridia had been solved. “The structure data seemed to suggest a new kind of activation mechanism for endolysins” Matt explains. “And it just looked interesting. You get to work on something that isn’t known before” he says enthusiastically. So it took several years rather than a few months, but now Matt, Rob and their collaborators have resolved the activation mechanism for two bacteriophages targeting two species of Clostridia bacteria – the known hospital bug Clostridium difficile (or C. diff) and C. tyrobutyricum which can cause problems in the cheese making process. Despite being two very different species, the structure of the part of the enzyme which carries out the activation mechanism seems very similar. “Even though the sequence similarity of this part of the enzyme is very low,” says Matt, “the structure similarity is relatively high”. If this means, as the group believe, that the mechanism is actually quite widespread, this information could open the way to developing technologies to fight a range of pathogenic bacteria in the health and food industries – not just Clostridia.
The problem with Clostridium difficile
Clostridia are gut bacteria. Clostridium difficile is becoming a major problem in hospitals. In healthy individuals it happily shares its environment with other gut microbes, but when patients are treated with broad spectrum antibiotics, this harmony becomes disturbed and the persistent C. diff. can increase uncontrollably in numbers, releasing toxins which cause severe cases of diarrhoea and also rarely life threatening inflammation of the intestine wall. Clostridium difficile is very hard to treat, being unresponsive to many antibiotics, and resistance is increasing. Some 250,000 C. diff infections are reported annually in the US, resulting in 14,000 deaths and $1billion of excess medical costs. In Germany the Robert Koch Institute reported a total of 1715 cases in 2013, an increase of 457 cases from 2012. Bacteriophage treatment could be a real alternative. In contrast to antibiotics, bacteriophages have the advantage of being very specific in what they attack. Rather than attacking whole communities of bacteria, with detrimental effects to the patients well-being, they will attack one species, sometimes even a single strain, without affecting other organisms.
Matt was thrown in at the deep end. ”The first 4 months of the PhD were a bit of a shock” Matt recalls. “Coming from a BSc I hadn’t done that much independent lab work before – but it didn’t take long to get myself established thanks to the great support and advice here in Hamburg” he says. The group started off using Small Angle X-ray Scattering with three different endolysins to try and observe the switch happening. ”We tried to induce the switch by changing the pH and salt concentration. It looked like we were getting a mixture of a mainly head on confirmation but also a side by side confirmation and some other combinations. So something was definitely happening”. After the compulsory introductory course at EMBL Heidelberg, Matt returned to the lab where he had exactly two and a half weeks with the group technical assistant who was about to go on maternity leave. “That was really intense”, he recalls, “she was the only one working on endolysins at the time, so she passed on everything. I still go back to the protocols she gave me – they are up here now”, he taps his head, “but that was a very useful time”. Matt carried on where she had left off. Since it looked like the endolysins were shifting from one configuration to another, the plan was to create mutants of the endolysins by playing around with the genetic sequence to try and understand how the switch from one configuration to another was working – the principle being if the created mutant is not active, the induced change has switched off the activity, and hence the residue required for activity can be deduced. “I think we did almost 30 mutants” says Matt. “We were really focussed on understanding what was going on”.
A bright future
That steep learning curve stood Matt in good stead and finally, using a mix of crystallography, small angle X-ray scattering and mutagenesis, he and his colleagues now believe that the switch from one confirmation to the other activates the endolysin which passes on through the cell membrane to destroy the cell wall. “Now we know how to use endolysins to treat pathogenic bacteria, we need to ensure effective targeting and increased activity. Several papers already show how just a few changes in the active site for example leads to increased activity”, Matt explains enthusiastically. “This is a huge area. The use of phages to treat foodborne pathogens is already on the increase. Phages are everywhere and they can be very specific, so they do not affect other organisms”. We don’t even have to use the whole phage, but exploit the bits that do the killing – that is why Matt’s results describing how these enzymes work is such an important breakthrough. “There is a whole range of applications using bacteriophage and their products such as endolysins” says Matt. “The future will tell what technologies are feasible and sustainable but I think there are seemingly endless opportunities and I’m sure we will find many useful ways of using them.” Technologies already exist, for example for the detection of even low levels of pathogenic bacteria such as Salmonella, E. Coli and Listeria in food products- an important tool in combating epidemics caused by food-borne pathogens such as the recent EHEC outbreak in Germany in 2011. And of course these findings bring a glimmer of hope into a pending “post antibiotic era” by giving us options for new ways of fighting pathogenic bacteria in the field of human health. But what about therapies for C.diff? “The problem is getting the endolysins down to where the bacteria are” says Matt. “If you just swallow them, they will be destroyed before they get there.” One possible solution could be to engineer a probiotic bacterial strain to secrete endolysin which would be added to a yogurt. Once inside the gut the bacteria inside the yoghurt could help to eradicate the C. diff bacteria by secreting the specific endolysin, leaving “good” gut bacteria untouched. So the future needn’t be quite so bleak after all, and I’m sure Matt’s future will be very bright.
Dunne, M., Mertens, H.D.T., Garefalaki, V., Jeffries, C.M., Thompson, A., Lemke, E.A., Svergun, D.M., Mayer, M.J., Narbad, A. & Meijers, R. The CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor. PLoS Pathogens, 24 July 2014.