Science Week 2017: Resistance – film screening and panel discussion

Dr Clare Sansom, Senior Associate Lecturer in Biological Sciences, writes on the screening of Resistance: not all germs are created equal and panel discussion on antibiotic resistance, which took place as part of Science Week 2017

resistance_panel-disc-3Antibiotic resistance is one of the most crucial issues facing humanity in the early 21st century, with some commentators even suggesting that it poses as serious a threat to civilization as climate change. It was therefore timely that one of Birkbeck Department of Biological Sciences’ contributions to Science Week 2017, with its strapline ‘Microbes in the Real World’, should tackle the issue. This took the form of a screening of an award-winning feature film from 2014, Resistance (subtitle: Not all germs are created equal) followed by an extensive and lively panel discussion. The four panellists were scientists from the department whose research is geared to the development of antimicrobial drugs: Dr Sanjib Bhakta, a Reader in microbiology; Professor Nicholas Keep, Executive Dean of the School of Science and a structural biologist; and two promising students from Dr Bhakta’s lab: PhD student Arundhati Maitra and MRes student Alina Chrzastek.

Not surprisingly, given the timeliness of the issue and (it has to be admitted) the size of the venue – the tiny Birkbeck Cinema in Gordon Square – the session was over-subscribed. After a short introduction by Dr Bhakta, who used his own research field of tuberculosis to set out the ‘global threat’ of drug resistance, the packed audience were treated to 70 minutes of engaging and at times chilling documentary. The film, by US producers Ernie Park and Michael Graziano or, collectively, Uji Films, uses a combination of archive footage, animation, interviews and personal stories to explain how we have arrived at a point where antibiotics are failing and what we need to do to ‘save antibiotics in order to save ourselves’. Although the film was made in the US and focuses on US policies and case studies, the problem it describes is a global one and it would not have been difficult to find equivalent examples in the UK.

The producers weaved three case studies of patients who had suffered antibiotic-resistant infections engagingly through the footage. We were introduced to a teenage lad who had been exceptionally lucky to survive drug-resistant pneumonia with some disability; a fit, middle-aged man who picked up methicillin-resistant Staphylococcus aureus (MRSA) while surfing and is now seriously disabled; and, most harrowingly, a mother whose 18-month-old baby picked up a new strain of MRSA and died within 24 hours.

The film’s narrators explained that all antibiotics are ‘poisons that kill bacteria but not us’; if they don’t kill the bacteria they make them stronger. Using antibiotics in such a way as to promote this rapidly sets up a ‘Darwinian battleground’ in which weak bacteria are knocked out but strong ones survive. This can happen very quickly because bacteria grow and divide so fast. In the words of scientist and author Maryn McKenna, we had the only effective way of killing bacterial pathogens and squandered it. And we have done this in three main ways: by over-use in the environment, in agriculture and in medicine.

The first two of these are particularly prevalent in the US and some Asian countries and less of a problem in Europe, where regulation is stronger. In the US, antimicrobials are used in everyday household products, sprayed on everything from fruit trees to kitchen counters. And once farmers had realised that constant small doses of antibiotics made livestock grow faster and fatter, even in crowded, unsanitary conditions, they were determined to keep doing so even though it ‘makes as much sense as sprinkling antibiotics on your children’s cereal’. Most US-produced meat and poultry is now contaminated with resistant bacteria, and occasionally this is multi-drug resistant. A Danish hog farmer, Kaj Munck, explained the sensible approach taken in Denmark where antibiotic growth promoters in animal feed were banned in 1995 following an extensive public debate. The Danish pig industry is still profitable, producing 28 million a year: about the same as the state of Iowa.

The beginning of the antibiotic era in human medicine coincided with World War II, when it was seen as a ‘miracle drug’ for curing infected wounds. Over-use, however, started very soon: penicillin was given to overseas sex workers, not to protect them from infection but to prevent their US military clients from becoming infected. The danger of resistance was known as early as 1945, when Sir Alexander Fleming told the New York Times that “in such cases the thoughtless person playing with penicillin is morally responsible for the death of the man who finally succumbs to infection.” Doctors who prescribe antibiotics inappropriately are often not morally wrong, or even thoughtless, but over-anxious to avoid mistakes when the chance of an infection being bacterial is low but not vanishingly so. Readily available, rapid diagnostic tests would go a long way towards preventing this type pf misuse.

It would not matter as much if antibiotics became ineffective if there were other molecules ready to take their places. However, the current antibiotic pipeline is weak, with few drugs coming through. Pharma companies can spend at least a decade and a billion dollars on developing a single drug, so it makes more sense to work on drugs like statins that patients must take every day. We must begin to encourage and reward companies that bring forward antibiotic ‘drugs of last resort’ rather than best-sellers. In short, the film concluded, the problem of antibiotic misuse is a classic example of ‘the tragedy of the commons’; one individual’s over-use of antibiotics may be neutral or even beneficial, but if everyone does it there will be a huge problem. To win the arms race against bacteria we may need to redesign all the processes through which we discover, use and protect antibiotics, and to ‘use our wits to keep up with their genes’.

Bhakta introduced the panel discussion with a short explanation of the molecular mechanisms through which bacteria acquire resistance to antibiotics. Bacteria evolve quickly, and almost all have acquired some resistance either intrinsically, through mutations, or by acquiring resistance genes directly from other species. This is an inevitable process but we have some control over how quickly it occurs: good antibiotic stewardship is as important as innovative science for winning the ‘arms race’ described in the film.

Bhakta’s group at Birkbeck is interested in tackling the problem of resistance through discovering new compounds with novel modes of action and by aiming to ‘re-purpose’ some over-the-counter medicines that are already in use for other indications. Drugs in this category will have already been shown to be safe and are therefore quicker and cheaper to develop. Keep summarised the role of structural biology in antibiotic discovery as one of determining the structure of bacterial proteins that might be vulnerable to attack by drugs and identifying compounds that can bind to and inhibit them. We are now often able to see directly how these structures are changed by mutations that increase (or decrease) resistance.

Bhakta chaired the discussion that followed, which was extensive and wide-ranging, taking in politics and economics as well as science and medicine. Several questions touched on the role and responsibilities of the pharmaceutical industry, which is reluctant to invest in drugs that will only be used for short periods. More drug discovery than ever before is taking place in academic labs and small companies, often working together; Maitra, whose Birkbeck Anniversary PhD studentship is part-funded by Wellcome, highlighted the role of the Trust in promoting links with industry. Re-purposing drugs that have already been used clinically is much cheaper than developing a molecule from scratch. MRes students in Bhakta’s lab, including Chrzastek, are testing common anti-inflammatory drugs against Mycobacterium tuberculosis and have found some potentially useful activity although the mechanism of action is still to be explored.

Other questions focused on the need for strict antibiotic control measures. In many European countries, including the UK, antibiotics are only available on prescription and cannot be used as growth promoters in animal feed. This ‘best practice’ needs to be replicated worldwide, but it will be an uphill struggle. Bhakta told the audience that he often visits countries in south and east Asia where resistance is prevalent and has seen antibiotics available over the counter there. In countries without strong, publicly-funded healthcare systems there are often incentives for doctors to over-prescribe drugs including antibiotics. And even where this is not an issue, patients need to be educated to think of antibiotics as drugs of last resort rather than demanding them for every upper respiratory tract infection.

It was perhaps inevitable that someone would ask the ‘Brexit question’: in this case, is there a danger that we would reverse some of our ‘best practices’ when we are no longer bound by EU regulations? Encouragingly, Bhakta doubted that anyone would want to get rid of rules with such clear benefits. He felt that the now inevitable move of the European Medicines Agency, which regulates all medicines marketed in the European Economic Area, from London – and the confusion about how the UK drug market will be regulated – does present a danger, to our strong research base. And however the politics develops the international collaborations that UK-based doctors, scientists and entrepreneurs have built up over decades must be maintained.

Other Science Week 2017 events:

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Protein machines in the molecular arms race

This post was contributed by Clare Sansom, Senior Associate Lecturer at the Department of Biological Sciences

Birkbeck’s Science Week 2015 was held from Monday 23 to Thursday 26 March and included three evenings of public talks by senior researchers. The first two lectures, on the Tuesday, were given by two of the college’s most distinguished women scientists: Helen Saibil from the Department of Biological Sciences and Karen Hudson-Edwards from Earth and Planetary Sciences; they were billed together as a ‘Women in Science Evening’.

Pathways of pore formation – illustration by Adrian HodelThe lectures were all introduced by the Dean of the Faculty of Science, Nicholas Keep who described Saibil, a close colleague, as “our most eminent female scientist”. She came to Birkbeck from Canada via a PhD at King’s College London under the supervision of Nobel laureate Maurice Wilkins and post-doctoral work at Oxford.

Since arriving here in the 1980s she has built up an internationally renowned structural biology lab, focusing in particular on the technique of electron microscopy. She has been a Fellow of the Royal Society since 2006 and of the Academy of Medical Sciences since 2009.

Saibil began her lecture by explaining that proteins can act as little machines, performing mechanical tasks that are essential for the maintenance of life. Her group has been interested for some time in proteins that can punch holes in the walls of cells. This allows the cell contents to leak out in a damaging process known as lysis, and it also allows toxins to enter the cells. These proteins can therefore be thought of as powerful weapons, and they are deployed on both sides of a ‘molecular arms race’: by pathogens and by the immune systems of humans and other animals.

Most soluble proteins fold into a single stable structure that tries, as far as possible, to keep their hydrophobic (“water-hating”) parts – the side chains of certain amino acids – in the interior of the protein, with the hydrophilic (“water-loving”) side chains on the outside, in contact with the watery environment inside or outside cells.

Pore-forming proteins, however, have a ‘Jekyll and Hyde’ like identity: they can form two distinctly different shapes, one as individual, soluble molecules and the other when they associate with each other into membrane-bound rings to form cylindrical pores. These structures, and the conformational change between them, are remarkably similar in proteins from bacteria and from the immune system.

Pore-forming toxins have been found in types of bacteria that are responsible for some deadly human diseases, including meningitis and pneumonia. The structure of a monomeric form of these proteins in solution was first solved in 1998, using X-ray crystallography. However, large complexes of many protein molecules are more readily solved by electron microscopy, particularly when those complexes are embedded in membranes.

In 2005 Saibil and her group described structures of the pore-forming toxin pneumolysin, from Streptococcus pneumoniae, in complex with a model cell membrane. They found that the proteins formed two distinctly different ring-shaped structures. Initially, they formed into a ring sitting on top of the membrane, which was termed the pre-pore; then they changed shape to burrow part of each protein deep into the membrane and form the pore itself. Each monomer in the pre-pore had a structure that was similar to that of the molecule in solution, but they underwent large structural changes to form the pore.

Most structures solved by electron microscopy are at lower resolution than those solved by X-ray crystallography, and it is not possible to trace the positions of individual atoms at lower resolutions (eg worse than 3 A). Saibil and her colleagues were able to interpret the structure of the proteins making up the pore by fitting pieces of the X-ray structure of the isolated molecule into their electron density.

They found a dramatic change in structure, with the tall, thin protein structure collapsing into an arch and a helical region stretching out to form a long, extended beta hairpin. It is these hairpins that join together to form the walls of the pore. The process of pore forming therefore has three stages: firstly the toxin molecules bind to the surface of their target cells, then they associate into the circular pre-pores and finally they change shape in a concerted manner, punching holes in the cell membranes by ejecting a disc of membrane, letting other toxins in and cell contents out.

Saibil then turned the focus of her talk from attack by bacteria to the human immune system’s defence. Natural killer (NK) cells are specialised lymphocytes (white blood cells) that kill virally-infected and cancerous cells in the bloodstream. They kill on contact with their target cells by releasing a toxic protein into those cells that stimulates those target cells to commit suicide in a process known as programmed cell death or apoptosis. We have only recently learned that the mechanism through which the NK cells work is very similar to the mechanism of the bacterial pore-forming toxins.

Natural killer cells express a protein called perforin that has a similar structure in solution to the bacterial pneumolysin. Although there is very little sequence similarity between these proteins – there is only one amino acid conserved throughout all the known bacterial and vertebrate proteins of this family, a glycine at a critical position for the conformational change – the structures are similar enough to suggest that the proteins all once had a common ancestor.

Saibil and her colleagues used electron microscopy to discover that this protein forms a pore through a similar mechanism to pneumolysin: the helical region that unfolds into the beta hairpin to form the pore forms the core of the molecular machine and is largely unchanged between the structures. There are some differences between the structures, however; in particular, there is no need for the perforin structure to ‘collapse’ as the molecule has ‘arms’ that are long enough to form the hairpin and punch the hole without bending into an arch.

The mechanism through which the NK cells kill their target cells is now quite well understood. When the two cells come into contact they form a temporary structure called an immune synapse that allows the pore to form and proteases called granzymes, which induce apoptosis, to enter the target cells. This YouTube video illustrates the natural killer cells’ mechanism of action, and this one shows a detailed view of the immune synapse. Other, similar proteins have been identified in oyster mushrooms; these form more rigid structures that are easier to work with. Saibil’s group and their collaborators have been able to solve the structure of this protein in intermediate stages of pore formation and are beginning to gain an understanding of exactly how it unfolds.

Mutations in perforin that prevent it from functioning cause a rare disease called haemophagocytic lymphohistiocytosis, which is almost invariably fatal in childhood. Understanding the mechanism of action of this important family of protein ‘weapons’ in both attack and defence may help find a cure for this devastating condition, as well as for some commoner disorders of the immune system and important infectious diseases.

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Image Caption: Pathways of pore formation – illustration by Adrian Hodel

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