With scientists on the alert for new therapeutics in the face of rising antibiotic resistance, the focus is shifting to specialised viral assassins that can demolish dangerous bacteria.
PlyC has been likened to a flying saucer, a doughnut and Pac-Man by scientists from the US and Australian research teams that have spent more than six years trying to take its picture – a crucial step if it is to be enlisted in the hunt for alternatives to antibiotics.
Found in a Milwaukee, US, sewer in 1925 where it was feasting on its meal of choice, Streptococcus, PlyC is a protein from a bacteriophage – a type of virus that has evolved over millions of years to kill bacteria, and with great specificity.
Bacteriophages, commonly called phages, were discovered in the early 20th century and quickly attracted the interest of doctors as potential therapies. They were used during World War II to treat battlefield infections.
But the discovery of penicillin, a drug that tackles more than one type of bacteria, dampened enthusiasm for these highly specialised viruses. Fast forward to present day, when the threat of a return to a pre-antibiotic era looms, and phages are again looking interesting.
King of the phages
Of all known phage proteins, PlyC – known as a ‘bacteriophage lysin’ – is king, acting at least 100 times faster than its peers to tackle strains of Streptococcus, the bacteria that cause throat infections, heart disease, pneumonia, toxic shock syndrome and topical skin infections in humans, as well as some animal diseases.
But to engineer any of these natural antibacterials into human therapies, researchers first have to know what they look like – a task involving vast amounts of complex research at a structural level.
“There are literally thousands of these types of molecules out there, all directed against different bacteria,” says Monash University’s Dr Sheena McGowan, lead author of the Proceedings of the National Academy of Science (PNAS) paper that revealed the structure of PlyC.
Dr McGowan and her Monash colleagues Professor James Whisstock and Associate Professor Ashley Buckle used X-ray crystallography to determine the structure of this molecule in 3-D. The team collaborated with Assistant Professor Daniel Nelson from the University of Maryland and Professor Vincent Fischetti from the Rockefeller University, in the US, both of whom have long histories researching PlyC.
“Penicillin and all of our antibiotics are magnificent drugs; however, we need to be thinking about a new generation of therapeutics,” Dr McGowan says. “With bacteriophages we’ve got a repertoire from nature. The idea is that if resistance over time started to build up against one of them, we have this whole range of others to choose from.”
In addition, because bacteria and viruses have been battling since the dawn of life, phages have naturally evolved to overcome new barriers of resistance in their bacterial hosts.
As the team found out, the power of PlyC is in its complicated structure: it has nine separate protein components. Its base is a ring with eight parts that dock to receptors on the cell wall of the streptococcal bug (other known phages have only one docking part). Two ‘catalytic sub-units’, the twin warheads, sitting on top of the ring then launch into action to chew through and ultimately destroy the bacterial cell wall.
“If you knock out one of the active sites the protein only has one per cent of its normal activity. But with both active sites, you have 100 per cent activity because they act in synergy,” Assistant Professor Nelson says. “It’s a monster.”
He started working on the molecule in 1999 as a postdoctoral researcher in the Rockefeller University laboratory of Professor Fischetti, the man who in 1968 purified PlyC from the phage sourced from the sewer four decades earlier.
The protein’s potency is evident from a video filmed by Rockefeller University researchers that compares two test tubes filled with milky streptococcal solution. A drop of PlyC solution is placed into one test tube. Within 30 seconds the liquid turns clear. At a microscopic level, PlyC has docked to the streptococcal cells, chewed through their walls and caused each cell to burst spectacularly.
“We have bacteriophages for many different bacteria – for Staphylococcus, for Streptococcus pneumoniae, for Listeria, for Bacillus, for Clostridium,” Assistant Professor Nelson says. “But this PlyC enzyme is 100 or 1000 times more active than any of the others. So if we can see why PlyC is so much more active, we can use that knowledge to make other enzymes like that.”
When Assistant Professor Nelson first joined Rockefeller as a postdoctoral fellow, he and Professor Fischetti quickly began talking therapeutics – topical throat sprays or lozenges with PlyC. In 2001, they published findings in PNAS that showed how a garden-variety throat infection cleared within hours of PlyC’s administration in animal models.
The efficacy was compelling but PlyC was coy about revealing its structure.
Coaxing crystals
Of the techniques used by molecular biologists to image the structures of potentially life-saving proteins, X-ray crystallography is one of the more dramatic, and one in which Monash excels.
At a conference in 2006, Assistant Professor Nelson got talking shop with Monash University’s Professor Whisstock, an Australian Research Council (ARC) Federation Fellow.
PlyC was soon on its way to the Monash laboratory of Professor Whisstock, who presented the structural puzzle to a postdoctoral researcher in his group, Dr McGowan, now an ARC Future Fellow.
Protein crystallography relies on a combination of X-ray technologies, computing power and the delicate art of encouraging the protein to form minute crystals.
“It starts in liquid form and you coax it to grow a crystal,” Dr McGowan says. “You want the crystal to fall out of the solution and become solid matter, but rather than lots of crystals, you need just one.”
To get the best crystal, crystallographers will try thousands of different chemical cocktails. Monash now has an advanced crystallisation facility – the Grollo Ruzzene Foundation Centre for Protein Structure – which uses robots to automate the crystallisation process. But back in 2006, Dr McGowan worked by hand.
The crystals are taken to a synchrotron, where X-ray beams are fired at each individual crystal. The diffracted X-ray patterns are processed by a computer to calculate the structure of the protein, which is then presented as an image.
Dr McGowan says the lower ring of PlyC practically imaged itself. “It was my first crystal structure and the data was of such high quality that we were able to use an automated process to determine the structure.” After a few days of number crunching, the Monash team was presented with a high-resolution saucer shape with a hole in the middle.
Secret in a shard
Growing a crystal that contained PlyC in its entirety turned out to be challenging, and at times seemed impossible. “I set up and tested more than 1500 crystals over two or three years,” says Dr McGowan, who thinks it was stubbornness, rather than patience, that carried her through the struggle with PlyC (a time during which she and Assistant Professor Nelson took over their own laboratories at Monash and the University of Maryland respectively).
“The crystals were stacked together and you can’t use them like that. The only way we ever got this particular data set was when a bit fell off and broke, and I picked it up. That was the only thing that ever diffracted to a resolution we could use.”
The data from this shard yielded nothing for a long time. It was only a last-ditch attempt in 2010 with a new computer program that provided Dr McGowan with the faint ghost of a helix, which, along with flat sheets, is one of the characteristic structures of a protein.
From this very fuzzy data set, Dr McGowan pieced together a 3-D jigsaw puzzle over the next 18 months, looking for “a corner to start from”. Working with multiple computer programs she built in somewhere close to 10,000 atoms manually, creating more helices and sheets. Every step of the way, the computer would verify whether what she was building worked from a mathematical perspective. If not, she would dismantle and start again.
Associate Professor Buckle, a National Health and Medical Research Council of Australia Senior Research Fellow, ultimately came in to help Dr McGowan connect these shapes, applying experience in chemistry and protein structure.
“We faced two difficult challenges. First, we had to piece together hundreds of small protein chain ‘fragments’ in a meaningful way – that is, to build something that looked like a protein,” Associate Professor Buckle says.
“Second, we had to assign the correct amino acids – the 20 protein building blocks – to the correct places. In reality you solve both problems simultaneously – reconnecting and re-sequencing, gradually improving the model as you go.”
This time-consuming and intense effort finally succeeded and the secret of the crystal shard was revealed: the business end of the molecule was a double-assault weapon.
Just why PlyC was so difficult to crystallise may be answered by an idea from Dr McGowan and Associate Professor Buckle – that the warheads change configuration before they start to chew the cell wall. The Monash researchers hope to build on the research to capture this unfolding process using computer simulation technology, providing a movie of the bacteriophage doing its dirty work.
But for the meantime, the photograph is enough.
