Wednesday, January 25, 2012

Driller killers: Turning bacteria's weapons on them

<i>(Image: Brian Larossa)</i>
(Image: Brian Larossa)

Bacteria battle each other with highly sophisticated smart impalers – now we’re turning this arsenal against them

MODERN weapons are becoming ever smarter. Armed drones patrol the skies of combat zones. Robot sentries are replacing humans. And some guns are designed to automatically detect and aim at potential targets. It is only a matter of time before fully automated weapons do battle on our behalf.
If the idea of robot warfare appals you, be grateful you're not a bacterium. In the battles that rage between rival groups, kamikaze bacteria burst open and spew out automated weapons to destroy their adversaries. Unlike us, they solved the targeting problem long ago - these deadly weapons kill only specific enemies. And it's not just other microbes that are under attack: some bacteria use these weapons to target big game, including mammals like us.
The good news is that we now know enough about these astoundingly sophisticated weapons to reprogramme them and turn them on our enemies. These weapons could help us tackle everything from deadly food poisoning bugs to insect pests - and they'll take out the bad guys without harming the good guys.
This story begins in 1954, when biologist Fran├žois Jacob at the Pasteur Institute in Paris, France, was studying Pseudomonas aeruginosa, a bacterium that today kills many people in hospitals. He discovered one strain released a substance that killed another strain of the same species.
That was somewhat unusual in itself. Most antibiotics kill a vast range of species, not specific strains. Later studies, though, confirmed that this substance - which Jacob dubbed a "pyocin" because P. aeruginosa was known as P. pyocyanea at the time - killed mainly specific strains of P. aeruginosa.
That was not the only curiosity. The pyocins were also extremely potent: a single particle could annihilate a bacterium.
Other researchers discovered that these pyocin particles are enormous compared with most of the toxins bacteria use to attack their foes. And when they looked at them under an electron microscope, they found they were very peculiar indeed. The particles resemble cartoon rockets, consisting of a thick tube with several fibres protruding from one end (see diagram).
While the particles might look like rockets, we now know they are actually a kind of cross between a floating mine and a spear gun. Inside the thick tube is another, narrower one. When the fibres grab hold of the surface of a cell, the outer tube contracts and propels the inner tube through the cell wall of its victim, quickly killing it. What's more, these are smart weapons. Since these "grabbers" latch on to specific strains of bacteria, the weapons kill only their designated targets, leaving other cells unharmed.

Smart impalers

P. aeruginosa is not the only bacterium that produces this kind of "smart impaler". It turns out that a range of bacteria make similar weapons. Some even use them to attack larger prey, such as insects.
The bacterium Serratia entomophila, for instance, attacks the beetle larvae known as New Zealand grass grubs (FEMS Microbiology Letters, vol 270, p 42). After infecting a grub, some bacteria release pyocin-like particles that latch on to the larvae's cells. The targeting is precise; the particles do not harm a closely related insect.
But these smart impalers kill in a more insidious way. Rather than merely making a hole in cells, they deliver a crippling toxin, says Mark Hurst, a molecular biologist at AgResearch in Christchurch, New Zealand. The details are still not clear but the result is: the grub stops eating. After a couple of months, when the starving grub has weakened, the bacteria swarm into its blood and begin devouring its tissues.
Smart impalers are powerful weapons, but there is a big downside for the bacteria that produce them. The components of smart impalers are made and assembled inside bacteria, but because they are so large the only way to release them is to break open the cell. In other words, releasing smart impalers is a suicide mission.
Why would some bacteria sacrifice themselves in this way? The simple answer is that strains of bacteria in which some individuals go kamikaze must have been more successful than those that don't. In fact, it is not that unusual for individual bacteria to sacrifice themselves "for the greater good". Far from being loners, bacteria are actually highly social organisms that often behave in ways reminiscent of the cells of multicellular plants and animals.
One bacterium, though, may not need to go kamikaze. Called Photorhabdus, it emits blue light and may have been responsible for the glowing wounds seen during the American civil war. Worms carry these microbes in their guts and spit them out after burrowing into an insect. The bacteria then launch an attack with a battery of weapons, including a kind of smart impaler. They liquify the insect's innards and set the corpse aglow as they feed on it.
Photorhabdus has machinery that may be capable of exporting the pieces of the impaler from the cell. So instead of exploding to release its smart impaler, says Nick Waterfield of the University of Bath, UK, the bacterium may assemble it outside its cell.
Like S. entomophila, Photorhabdus produces poisonous impalers. When Waterfield's team studied them, they found a toxin near the ends of contracted ones, as if it had been squirted out, he says. Genetic studies suggest that Photorhabdus can load its smart darts with any one of a variety of toxins, and Waterfield suspects that the different toxins may kill different insects.
The poisonous impalers are certainly highly effective, as Waterfield's team found when they injected them into wax moth larvae (Journal of Bacteriology, vol 188, p 2254). "It was totally lethal," he says. "They went black and died within 15 minutes."
Other bacteria avoid sacrificing themselves by taking a cruder approach. Instead of releasing smart impalers, they engage in hand-to-hand combat. In these bacteria, impalers stick out of the cell wall and stab other cells the bacterium comes into contact with. Some have poisoned tips or inject toxins into cells, and they can even kill mammalian cells. A recent study found that a close relative of the cholera bacterium can kill predatory amoebas and mouse cells with its built-in impalers.
These stabbing weapons - known to biologists as type VI secretion systems - may come in handy when this bacterium infects animals. Some biologists suspect that after it enters a human intestine, it stabs and kills other bacteria to make room for itself. And when our immune cells attack these bacteria, they may allow themselves to be swallowed and launch a Trojan horse-style attack after they get inside the cell.
So how did bacteria acquire such extraordinary weaponry? Many of the smart impalers have a striking resemblance to the viruses that attack bacteria, known as bacteriophages. These viruses have a capsule, or head, containing their genetic material, attached to a tube with grabbers on the end. They latch on to specific bacteria and inject their genes into them. The infected bacterium is forced to make lots more viruses and eventually bursts open to release them.
The auto-impaler discovered by Jacob, now known as an R-type pyocin, looks just like a so-called contractile-tailed phage minus its capsule. The type VI secretion system also looks just like a contractile-tailed phage, but minus both the capsule and grabbers. Another kind of pyocin with a more flexible tube, named F-type, looks just like a lambda phage minus its capsule.
The resemblance is so close in these cases that most biologists believe it is unlikely that these structures evolved independently. On several occasions, the genes coding for a bacteriophage may have become incorporated into the genome of a bacterium. Mutations in these genes then produced impalers like R-type pyocins. "Basically, the head was chopped off," says Ry Young, a phage biologist at Texas A&M University in College Station.
But what makes pyocins so lethal, given that phages do not kill their victims until their offspring are ready to burst out of its body? One idea is that phages somehow seal the hole they make in their victims.

Old weapons, new targets

However impalers evolved, their similarity to phages could turn out to be extremely useful. For nearly every bacterium, there is a phage that infects it. Researchers at AvidBiotics of South San Francisco, California, realised that by swapping the grabbers on a pyocin with those of a phage, they might be able to produce smart impalers that target just about any bacterium. Unlike antibiotics, which kill relatively indiscriminately, treating infections with smart impalers would leave helpful bacteria unscathed. "They're almost like a magic bullet," says Dean Scholl, a microbiologist at AvidBiotics.
The team decided to give it a try. They replaced the DNA coding for the grabber of a pyocin with DNA coding for a hybrid grabber whose business end came from a phage that kills many strains of the bacterium E. coli. Just as the researchers hoped, the engineered smart impaler killed E. coli instead of its previous targets, they reported in 2008.
Next, the team engineered a smart impaler to kill a particularly nasty strain of E. coli called O157:H7. When the pyocin was given to infected rabbits, it prevented or reduced symptoms such as diarrhoea (Antimicrobial Agents and Chemotherapy, vol 55, p 5469).
AvidBiotics has also made pyocins that target Salmonella, Shigella, and the E. coli strain that caused the 2011 outbreak in Germany, says Scholl, and it is working on pyocins against the cholera bacterium. The company is focusing chiefly on gut infections because if foreign molecules like smart impalers were injected into our bodies, they would trigger an immune response.
That means repeated injections would rapidly become ineffective, because our bodies would mop up the impalers. In theory, it might be possible to alter the structure of impalers to avoid immune attack, but this is a major undertaking that no one is attempting at the moment.
Besides gut infections, pyocins might also prove effective against lung infections, as inhaled auto-impalers may have a chance to kill bacteria before getting mopped up. Another use would be to kill bacteria on food. Ecolab of St Paul, Minnesota, is working out how to manufacture large quantities of the E. coli O157:H7 pyocin and the best way to apply it to beef. It aims to release the product later this year, according to John Hilgren, a microbiologist at the company.
Bacteria can evolve resistance to smart impalers, as they do with normal antibiotics. AvidBiotics found some E. coli O157:H7 bacteria evolved to evade attack by getting rid of the sugary coat to which the engineered pyocin binds. But since the sugars protect the cells and help them stick to human guts, the sugar-free bacteria were less dangerous, suggesting pyocin-resistance may not be a major problem.
Meanwhile, insect-killing smart impalers could prove useful in agriculture. Farmers already use S. entomophila to control New Zealand grass grubs, which eat the roots of grass. But it can take a few days for a grub to stop eating after it is infected with the bacteria. Purified impalers halt the grub's feeding overnight, so they should be even more effective, says Hurst.
Some biologists even hope to turn smart impalers into nano-syringes for delivering drugs to cells. Waterfield's team is trying to work out how toxins are packaged into the smart impalers produced by Photorhabdus. In theory, one could load other molecules into the auto-impalers and make them target specific kinds of human cells, he says.
With their precise targeting and ruthless efficiency, auto-impalers are nearly perfect nanoweapons. And now we're the ones choosing their targets.

Set phages to kill

Smart weapons that kill dangerous bacteria can be engineered by using parts of the viruses that attack them (see main story), but why engineer new kinds of treatments at all? Why not just use the live viruses, known as bacteriophages?
Some doctors do. The first attempts to treat bacterial infections with phages were made about a century ago, and phages are still used as medical treatments today in the former Soviet state of Georgia. But phage therapy never caught on in the west, although the viruses are used to kill bacteria in some food-processing facilities.
Part of the problem with using phages is that they change and evolve. And sometimes they genetically modify bacteria rather than killing them. The E. coli strain that made hundreds of people seriously ill in Germany last year, for instance, acquired the genes that made it so dangerous from a phage.
This is why, rather than deploy living phages, researchers are raiding the phages' arsenal of weapons in search of more dependable ways of killing bacteria. One such weapon is a protein called a lysin, which phages use to bust out of host cells. The lysin drills through the cell wall of bacteria, making them burst open. Lysins can also kill some bacteria from the outside.
In 2010, microbiologist Vincent Fischetti and his colleagues at The Rockefeller University in New York reported that an engineered lysin killed the superbug MRSA on mouse skin more efficiently than an antibiotic. ContraFect of Yonkers, New York, hopes to make products for tackling several pathogens, including MRSA and anthrax-causing bacteria, based on Fischetti's work. Another team at the US Department of Agriculture in Beltsville, Maryland, is creating chimeric lysins that hit three parts of the cell wall at once, making it very hard for bacteria to evolve resistance.

Roberta Kwok is a freelance writer based in the San Francisco Bay area

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