Structural and molecular investigations of membrane-embedded pilus assembly nanomachines in Gram-negative bacterial pathogens

Bacterial pili are cell surface appendages produced by bacterial pathogens. Pili are used by bacteria for recognition of and attachment to host tissues. Once attached to the host, the bacterium can start the process of infection. Therefore, pili are important means by which a pathogenic bacterium infects a host.
Pili are large polymers of protein subunits termed pilins. Polymerisation of pilins into a mature pilus is mediated and orchestrated by macromolecular complexes embedded in the bacterial cell envelope. These complexes are large and often composed of many proteins. They function like machines and catalysts, grabbing individual pilins, catalysing their polymerisation, and facilitating their secretion through the bacterial envelope and their exposure at the bacterial cell surface. The major objective of this proposal is to investigate the structure and function of these machines, to understand how they work, and also, eventually, to find ways to inhibit them.
Inhibiting the production of pili by bacteria would be very advantageous: i- without pili, the pathogenic bacterium cannot attach to its target tissue, and is therefore harmless; ii- because selective inhibition of pilus production is non-lethal, all the "good" bacteria which we naturally harbour in our bodies (the microbiome) and which play very beneficial roles will survive treatment. The concept of only inhibiting the pathogen while protecting the "good" bacteria from antibiotics action is a new concept in antimicrobials design, a concept that holds enormous promise for combating infectious diseases caused by bacteria.
We are in a unique position to achieve these goals. Our track record is unique in the field of bacterial pathogenesis in general and pilus biogenesis in particular. Our structural biology work can be directly used to design small molecules capable of binding and disrupting crucial steps in the process of generating pili and secreting them.

This proposal aims to understand the mechanism of pilus assembly by two systems: the chaperone-usher (CU) pilus and the type IV pilus (TFP) assembly systems. Both systems are of significant biomedical importance: CU pili have been intensely characterised in uropathogenic E. coli, where they play well defined roles in host tissue recognition and adhesion; the role of TFP pili has been characterised in a number of bacterial pathogens including Neisseria meningitis, the TFP system of which we propose to study.
Our research programme aims to determine the structure of these assembly systems and carry out a detailed biochemical and biophysical characterisation of their mechanism. To carry out the proposed research, we will use a multidisciplinary approach combining molecular biological, biochemical, and biophysical methods such as X-ray crystallography, and cryo Electron Microscopy (EM) to gain structural information, and fluorescence resonance energy transfer (FRET) to gain dynamic information on domain/protein motions during pilus biogenesis.
For the CU pilus assembly system, we have derived a general model for system-mediated pilus biogenesis, which we propose here to investigate by solving additional structures to further characterise the properties of the system, and by fluorescence spectroscopy to analyse the details of protein and domain motions during pilus assembly. For the TFP assembly system, which is at a less advanced stage of structural knowledge, we will first determine the structure of the assembly system either by EM or X-ray crystallography. Next, we will study the recruitment of pilus subunits to the assembly system. Once these two objectives are fulfilled, we will aim to investigate the detailed mechanism of TFP biogenesis using solution biophysics methods similar to the ones used to dissect the mechanism of CU pilus assembly.
In both cases, we will seek to exploit our detailed knowledge to design inhibitors of pilus biogenesis.

The planned research will first benefit the larger public. Our research programme has already produced compounds that are effective in inhibiting pilus biogenesis by the chaperone-usher pilus assembly machinery. A patent on this first series of compounds has been filed. Recently, we have generated a second series of compounds, very different from the first ones and targeting a very different step of the assembly mechanism. A patent on this latest generation of compounds is in the process of being filed. This demonstrates that our research is effective in generating effective Intellectual Property that can be further exploited. Our translational activities are at the leading edge of a new field in antibiotics research, that of antivirulence antibiotics, i.e. compounds that target specifically virulence factors in view of disabling the bacterial pathogen. Indeed, after 60 years of tremendously successful research which has resulted in an arsenal of very effective antibiotic compounds, resistance to these compounds is rising. We are now at a threshold of a very dangerous public health crisis, notably in hospitals where untreatable nosocomial infections are on the ascent. Yet, finding novel antibiotics has proved difficult and many major pharmaceutical companies have withdrawn from the field. This state of affairs combined with major breakthroughs in understanding the mechanisms of bacterial infections has led to questioning the traditional model of the past. Indeed, all antibiotics kill the pathogens and as a result, as soon as an antibiotic is on the market, resistance appears. Moreover, all antibiotics wipe out the commensal bacterial population that inhabits humans, and side effects of this are only recently being recognised. Indeed, the bacterial microbiota represents 90% of the human cell population (in number) and has now been demonstrated to be responsible for a number of metabolic and immune diseases such as obesity and asthma. It is now clear that antibiotics, by killing both the good (commensal microbiota) and the bad (pathogens) have unintended consequences. It is therefore time to move to a different approach in antibiotics design: the targeting of virulence factors. Disarming the bacterial pathogen instead of killing it is a viable alternative. It has the advantage of i) lowering considerably the selective pressure for resistance and ii) preserving the microbiota intact. Elimination of the disarmed pathogen is then completed by the immune system. This is a novel approach that breaks from the traditional bacterial killing approach.
The planned research will also benefit society as a whole by creating wealth through the development of economic activities. Indeed, in the relatively short term, our continuing effort in antivirulence drug design should result in clinical applications which we hope will be wealth-creating, either through licensing to existing companies or through founding our own.
Finally, the proposed research will contribute to the training of talented scientists. I have been continuously funded by MRC from the time I moved my laboratory from the USA (2002). Since then and over a period of 10 years, I have trained or am in the process of training 21 postdoctoral associates and 11 PhD students. Except for 2 individuals who have left research, all have obtained positions in either academia or industry. Thus, the training provided in my laboratory is effective in providing the highest training standards requested from both Academia and Industry in the UK.