Newsletter - Edition 27

New Targets in the Fight Against Antibiotic Resistance

Oxford Innovation Society Lecture December 1998
Professor Jeff Errington, Sir William Dunn School of Pathology, University of Oxford

In the last few years there has been intense media interest in the problem of antibiotic resistance in bacteria. Few would dispute that there is an urgent need to develop new classes of antibiotics and most big pharmaceutical companies are investing heavily in this area. My lab has been interested in the molecular biology of bacteria for many years, but mainly in problems far removed from antibiotics and resistance. However, recent progress in understanding some fundamental aspects of bacterial structure and function have opened up possible routes to the development of new antibiotics. In this lecture I have tried to give a flavour of the excitement that has been generated in my field of research in the last few years, mainly by the application of new microscopic methods. I then gave a brief description of how we intend to exploit this opportunity commercially, through our new spin-out company MicroGenics Ltd.

To set the scene, it is necessary to give a brief introduction to bacteria (sometimes called prokaryotes) and how they differ from cells of higher organisms (eukaryotes). Firstly, bacteria are very small, typically only about 1 or 2 mm in length. Many thousands of such cells could fit inside a typical human cell. Bacterial cells also appear (at least superficially) to have a much simpler organisation than human cells. In particular, they generally lack intracellular membrane-bounded organelles, such as mitochondria, lysozomes, endoplasmic reticulum, Golgi apparatus, etc. Even their DNA appears disorganised with no enveloping nuclear membrane. Traditionally, the shape of the bacterial cell is determined by a rigid cell wall lying outside the cytoplasmic membrane. There seemed no obvious counterpart of the cytoskeleton (proteinaceous microtubules and filaments) that controls the sometimes complex and variable shape of eukaryotic cells. However, this view of a simple subcellular organisation in bacterial cells turns out to be wrong. New discoveries about proteins involved in cell shape determination and cell division are dramatically changing our view of bacterial structure and function. As we shall see, these discoveries may have implications for the fight against antibiotic resistance by providing a range of new targets for us to attack.

My lab has worked on a particular bacterium called Bacillus subtilis for many years. It is an important industrial organism, being responsible for the bulk of the world's industrial enzymes (proteases, amylases, etc.), which it manufactures and secretes very efficiently. It is also an excellent experimental organism, easy and quick to grow, and very safe, actually being eaten in natto, a kind of yoghurt, in the Far East (though B. subtilis related to important pathogens). One of the most attractive properties of B. subtilis is its ability to take up DNA from the environment. This makes it extremely amenable to manipulation and study by molecular genetic methods. Finally, it has a very interesting life cycle, in which it forms a specialised cell type called a spore by a simple developmental process.

Interest in the developmental cycle of B. subtilis was an important factor driving the recent development of new microscopic methods that allow us to see the localization of proteins inside bacterial cells. However, these powerful methods have turned out to be extremely informative when applied to a range of problems of bacterial cellular organisation and have brought about a revolutionary change in this field. One particularly versatile tool is an intrinsically fluorescent protein called GFP (green fluorescent protein), which comes from a jellyfish (Aequorea victoria). GFP can be readily fused to B. subtilis genes by standard genetic engineering methods.

The hybrid protein then carries a fluorescent tag that can be visualised in fixed or even living bacterial cells by fluorescence microscopy. Related methods involve use of fluorescently labelled antibodies to detect the target protein.

Many of the most important advances based on these new methods have been in the area of cell division. Each time a cell divides it needs to replicate its genetic material (DNA) and move the two daughter molecules away from each other. A new wall then needs to be built between the DNA molecules to separate the rest of the cell constituents into two parts. Until recently, little was known about the mechanisms of cell division in bacteria. When B. subtilis sporulates, it uses an unusual, asymmetric form of cell division, and some properties of this process have turned out to be very informative about the general mechanisms of cell division.

I have chosed three particular problems from ongoing work in the Errington lab to illustrate some of the exciting developments in this field. The first area concerns the nature of the machinery involved in splitting the cell into two parts. A combination of molecular genetics and fluorescence microscopic methods has revealed that at least 10 different proteins participate in the actual division process. These proteins assemble, more or less in sequence, to form a circumferential ring around the middle of the cell (the "Z ring", because its major component is a protein called FtsZ). Some components of the Z ring have a mechanical role, driving constriction of the cell membrane. Other proteins contribute as enzymes, filling the new septum with rigid wall material. In this way, each of the new daughter cells will have a complete, intact cell envelope. Many of the details of assembly and constriction of the Z ring are yet to be resolved, but identifying the components and finding that they all assemble into a ring-like machine at the site of division represents a major step forward.

One of the interesting problems that follows from the above work is the question of how the Z ring is assembled precisely at mid cell. Although a complete answer is again not yet available, some components of this mechanism have been uncovered. Important clues have come from experiments with mutant cells deficient in the function of genes called min. When a cell divides, two new cell poles are formed at the site of division. Cells lacking one of the min genes often aberrantly divide again at the site of a recent division, to produce a tiny cell with no DNA called a "minicell". The min genes therefore provide bacterial cells with a system that prevents the division machinery from remaining active at the site of a recent division. Localization of the Min proteins encoded by these genes has revealed the simple but elegant mechanism that these proteins use. When cells divide they accumulate the Min proteins in the new cell poles. These proteins are antagonists of division, which prevent subsequent assembly of the division apparatus in their vicinity. At the same time, restricting the inhibitory Min proteins to the cell poles ensures that division can go ahead in the middle of the cell. The third problem discussed concerns the segregation of chromosomes. The decades-old view was that bacterial chromosomes are attached to cell membrane and that the newly replicated sister chromosomes are pulled apart gradually in parallel with growth of the cells. We now know that very active mechanisms are involved. A breakthrough came from studies of sporulation, when a graduate student in my lab, Ling Juan Wu, noticed that cells of a mutant of B. subtilis didn't segregate its chromosomes properly during sporulation. Eventually we found that a protein called SpoIIIE catalyses a remarkable reaction in which almost a whole chromosome is transferred through a small pore in the division septum. Although this reaction initially appeared to be highly unusual, the protein is highly conserved throughout all manner of bacteria. Its general role in non-sporulating bacteria probably lies in helping cells to recover from the potentially catastrophic consequences of trapping their DNA in the division septum. SpoIIIE blocks the last steps in septum closure and pumps the DNA out into one of the cells, allowing completion of cell division and survival of both progeny cells. Work on SpoIIIE and sporulation eventually led to the discovery of another protein, Spo0J, which is involved in actively pushing sister chromosomes apart before cell division. Again, this protein is conserved across almost all bacterial species, and its discovery and properties have had an important impact on our view of bacterial organisation.

Having illustrated some highlights of recent work on cell division in bacteria, how is this relevant to industry? To explain, it is first necessary to consider how antibiotics work. The first property an antibiotic needs is toxicity - the drug needs to be able to kill or severely impair the growth of bacteria. To do this the chemical usually acts to inhibit a specific bacterial function, or group of functions, that is essential to the bacterium. The second crucial property of antibiotics is selectivity - they must have little or no toxicity for the patient. To achieve this, the lethal target of the antibiotic should ideally be one which is not present in humans. The number of "targets" on which existing antibiotics act is surprisingly limited. However, the recent exciting developments in "bacterial genomics" have provided complete catalogues of bacterial genes. By finding out which genes are essential for the bacterium and knowing which are absent from humans, a greatly expanded list of potential antibiotic targets has been generated.

The main challenge now for industry is to find ways of looking for chemicals that will act on these new targets. To do this, it is generally crucial to have some idea of the function of the target. This is where Universities have an important role, because it is here that fundamental knowledge about the functioning of bacterial genes is most likely to be generated. MicroGenics Ltd was founded in June 1998 in a collaboration between the University of Oxford (Isis Innovation Ltd) and the Oxford-based pharmaceutical software company, Oxford Molecular Group plc. It will initially exploit novel methods stemming from fundamental research in the Errington lab to find potential antibiotics acting on a number of targets involved in bacterial cell division. Through partnerships with other members of the Oxford Molecular Group, MicroGenics will offer a range of services from consultancy and licensing of the assays, through to projects to develop lead compounds for clinical trials. The next few years promises to be a very exciting period now that the needs of industry have converged with those of basic research.

Newsletter - Edition 27 Contents

1. Antibiotic Resistance
2. From Lab to Ltd Company
3. Protein Misfolding and Aggregation