Science: On the trail of the resistance

Researchers at Leeds University have fresh hope of winning the war between antibiotics and bacteria. By Simon Hadlington

An international team announced yesterday that it has sequenced the genome for Mycobacterium tuberculosis - known otherwise as TB, the cause of millions of deaths every year.

It may have come just in time: strains of TB resistant to antibiotics are on the increase, aided by people unable or unwilling to complete courses of the drugs that could kill the bacteria.

But that's only part of the picture. The British government will shortly publish a major review of the problem of bacterial resistance to antibiotics. It will echo the findings of the House of Lords science and technology committee, which in April warned that the country was in danger of returning to the pre-antibiotic era, with many diseases becoming untreatable.

This doesn't surprise Ian Chopra, professor of microbiology at Leeds University and research director of its antimicrobial research centre, where scientists pool their expertise to identify antibiotic compounds and other therapeutic approaches.

"There is a lot of talk about the misuse of antibiotics, but it is inescapable that, if you use an antibiotic, you will create pressure for the selection of resistant organisms," says Professor Chopra. "There has been a sense of complacency about this. Many pharmaceutical companies had given up on antibiotics to concentrate on drugs for cancer or diseases of the central nervous system. But now that antibiotic resistance is beginning to have an impact, a number of large companies have reactivated antibiotic research programmes."

The problem is that evolution has equipped bacteria with a means to cope with virtually anything that we can throw at them. It's a structure called a plasmid.

As with all living cells, a bacterium's genetic information is contained in the chromosome in the form of DNA. However, bacteria also have small loops of DNA that are separate from the main chromosome. Called plasmids, these are at the heart of the organisms' ability to outwit the antibiotic.

"Plasmids don't generally contain the housekeeping genes necessary for the day-to-day survival of the cell," says Professor Chopra. "Rather they contain 'back-up' genes that might one day be needed in an emergency, such as survival when confronted by an antibiotic."

Antibiotics kill bacteria in a variety of ways. The beta-lactam class of antibiotics, which includes the penicillins, act by interfering with the synthesis of the bacterium's cell wall.

The sulphonamides inhibit the synthesis of bacterial DNA, while other drugs, notably a relatively new class called the fluoroquinolones, block the replication of DNA. Others such as chloramphenicol and tetracycline disturb the synthesis of proteins in the bacterial cell.

However, antibiotic resistance is a reality. Genes can move between the chromosome and the plasmid, and plasmids can move relatively freely between organisms. If an organism spontaneously develops or already has a gene conferring resistance to a given drug, that gene can be passed around to other cells and incorporated into their primary genetic apparatus. "Once an organism has become resistant, the resistance will very rapidly establish itself," says Professor Chopra.

So the challenge is to develop new ways to kill bacteria. The Leeds researchers are especially interested in the "rational" design of new drugs. This involves identifying potential targets in the bacterial cell - usually proteins - elucidating in fine detail the 3-D structure of the target and then designing small molecules that could interfere with the function of the target.

"This whole process is being enhanced by the large-scale bacterial genome sequencing projects," says Professor Chopra. "We already have

the complete gene sequences of several pathogenic bacteria and soon will have them all. We will be able to knock out a particular gene to see if it kills the bug. If it does you can clone the gene, isolate the protein it encodes and get the chemists to model an appropriate inhibitor."

Simon Phillips, professor of biophysics at the research centre, uses X-ray crystallography (the same technique that unravelled DNA's structure) to work out the 3-D shape of large protein molecules. X-rays focused on crystals of the sample protein are scattered by its atoms; a computer then decodes the pattern of scattering to suggest how the atoms are arranged in space. Professor Phillips's team has succeeded in crystallising an enzyme that is responsible for resistance to one of the early antibiotics, thiostrepton, which works by binding to the cell's machinery for synthesising proteins.

However, mutant bacteria developed an enzyme that inserted an obstacle where the thiostrepton molecule fitted - like squirting superglue into a keyhole. "The enzyme is called a methylase, and we have made crystals of it," says Professor Phillips. "By finding its 3-D structure it may be possible to devise a way of disabling it. Thiostrepton could then be resurrected as an antibiotic if it was administered with the methylase inhibitor."

Meanwhile, Professor Peter Henderson and his colleague Dr Richard Herbert are focusing on how bacterial cells eject antibiotics.

The membrane that surrounds cells contains proteins that can transport nutrients into the cell or throw out unwanted substances. The 3-D structure of such transport proteins is crucial to their function, yet none has so far been elucidated.

In some organisms, resistance arises because the drug is "effluxed" by transport proteins - the cellular equivalent of being turfed out of a nightclub by a bouncer.

Dr Herbert is using nuclear magnetic resonance to determine the structure of membrane transport proteins. "Once you have the structure of the site on the protein that binds the antibiotic you can postulate that, if you change the shape of the antibiotic here or there, it will no longer be effluxed, and that mechanism of resistance will disappear."

The superbug MRSA (methycillin-resistant staphylococcus aureus) was susceptible to a drug called norfloxacin before it developed resistance. A close look at the cell's membrane revealed a protein called NorA that could kick norfloxacin out of the cell and may be responsible for its resistance.

Using genetic engineering techniques, Professor Henderson's team has grown large quantities of a closely related membrane protein to NorA, which they are now attempting to crystallise. "The Holy Grail is to develop an inhibitor for these efflux proteins," says Professor Henderson. "This would allow us to resurrect older antibiotics, using them in conjunction with something that will disable the 'molecular bouncer'."

Certainly, something is needed soon. No new antibiotics have been discovered since the 1970s. One approach or the other needs to work.

Yet it would be ironic if, after all the effort that has gone into the sequencing of bacterial genomes and teasing out their gene sequences (which in TB consists of about 4,000 genes), the answers were provided by the technology that kicked this revolution off - X-ray crystallography and the understanding of 3-D structure, rather than the "flat" understanding that gene collections offer.