Characterisation of disulfide bond formation in bacterial pathogens: Unravelling the adaptation of a classical pathway into a virulence aid.

Bacteria are fascinating organisms that are able to adapt to a variety of environmental conditions. However, because of this characteristic, bacterial pathogens are able to quickly evolve to bypass human antibacterial measures. The rapid increase in resistance to antibiotics, combined with the slowing to a trickle of new antibiotics progressing through the pipeline over the past decades, could soon lead to a public health crisis.

A major reason for antibiotic resistance development is the fact that current antibiotics target components of the bacterial cell (usually proteins) which are essential for the viability of the microorganism. This creates selective pressure for the survival of bacteria that are resistant to the antibiotic in use. A way to avoid the emergence of 'superbugs' is to try to render pathogenic bacteria harmless by developing compounds that target their weaponry i.e. the molecules that allow bacteria to invade and damage their host. In this context, I am interested in investigating the option of using a central bacterial pathway, which is involved in the assembly of these weapons, against bacterial pathogens.

In bacteria, the Disulfide bond (DSB) protein system is responsible for the formation of additional linkages (called disulfide bonds) in proteins which are located outside the main cellular compartment and need to withstand harsh environments. Since the majority of molecules that promote bacterial virulence are protein-based and are also located near the outer surface of the cell, they are dependent on the DSB protein system for correct assembly. Therefore, by studying the DSB proteins of pathogenic bacteria we will eventually be able to use this knowledge for developing new, efficient ways of neutralising bacterial disease-causing organisms without promoting antibiotic resistance.

My hypothesis is that in bacterial pathogens the DSB pathway has diversified, compared to organisms that are harmless. This allows pathogenic bacteria to optimise their toolkit for invading the host and evading the host's defence mechanisms. My research project consists of three parts all related to this hypothesis. In the first part, I aim to use bioinformatics to determine all variations of the DSB pathway in pathogens and to identify underlying common traits between DSB protein systems of pathogenic bacteria. This is the first step towards developing novel approaches against bacterial virulence. In the second part of my research project, I will study a pivotal component of the DSB system (a protein called DsbA, which is essential for the assembly of several bacterial weapons) in the human pathogen Neisseria meningitidis, the causative agent of meningitis. In this bacterium there are three copies of DsbA and their function remains unclear. By elucidating their involvement in the pathogenesis of N. meningitidis we can find additional ways to combat this pathogen in the future. The third and final part of my research project will focus on the extremely antibiotic-resistant bacterium Pseudomonas aeruginosa which causes severe problems in post-operational and immunocompromised patients. I plan to study the two copies of the protein DsbD, which are found in this organism and are thought to contribute to its virulence with the scope of contributing to the design of attenuated bacterial strains that could be used for vaccine development.

My proposed research aims to acquire fundamental knowledge about the role of a central protein system in bacterial pathogens. This knowledge is key for the development of much-needed new antibacterial strategies which will prevent the emergence of antibiotic resistance in the future.

In Gram-negative bacteria the Disulfide bond (DSB) system is the master regulator of disulfide bond formation, a process which is required for extracytoplasmic proteins to fold and operate. It also has a prominent role in the pathogenesis of Gram-negative bacteria as it is involved in the assembly of numerous virulence factors and it is essential for processes like adhesion, toxin/enzyme secretion, capsule biogenesis etc. I aim to explore the role of this protein pathway in mechanisms of bacterial pathogenesis, with the long-term goal of contributing to the development of novel antibacterial strategies. My proposed research will combine a comprehensive bioinformatic approach with experiments on the DSB systems of two human Gram-negative bacterial pathogens, Neisseria meningitidis and Pseudomonas aeruginosa.

The DSB systems of pathogenic bacteria often diverge from the classical paradigm by containing more than one analogue of key DSB proteins. I will use bioinformatics to identify the full set of analogues of each DSB protein across all Gram-negative bacteria. This will allow me to pinpoint underlying common traits between the DSB systems of pathogens and to understand the evolution of this pathway into a virulence aid. I will work on N. meningitidis, which encodes three analogues of the oxidase DsbA, in order to elucidate the contribution of these proteins to the assembly of virulence factors. I will identify the full set of DsbA substrates via, amongst other in vivo techniques, differential proteomics. Finally I will use P. aeruginosa, a pathogen that encodes two analogues of the reductase DsbD, to understand the connection between this duplication and the protection of the bacterium against oxidative stress. I will also crystallise and determine the structure of the unique transmembrane reductant conductor DsbD. These studies could offer new ways of developing vaccines against a multi-drug resistant pathogen like P. aeruginosa.

The ever-increasing resistance of bacterial pathogens to currently used antibiotics requires our urgent attention. Gram-negative bacteria, especially, pose significant financial stress to health systems not only because they are resistant to almost all available antibiotics, but also because none of the newer classes of compounds is effective against them. It is absolutely essential that we develop new antibacterial strategies to combat bacterial pathogens but also to avoid encountering the same problem in the future. For this reason every available route towards this goal should be explored and is likely to affect the quality of life of future generations.

The proposed project is in line with the promising strategy of targeting bacterial virulence which will lead to the production of new, effective antibiotics and to the avoidance of bacterial antibiotic resistance. The Disulfide bond (DSB) system is a central pathway in Gram-negative bacteria which is intimately connected to bacterial virulence. Elucidating this connection will lead to the identification of novel ways of neutralising pathogenic bacteria in the future. The combination of a broad bioinformatics part with two focused experimental parts ensures potential long- and short-term contributions to the battle against bacterial pathogens. The identification of all DSB protein analogues across Gram-negative bacteria (resulting from the bioinformatics part of the research plan) will generate a complete image of the distribution of DSB proteins in pathogens and will allow the identification of underlying common traits that could eventually be used to identify new targets and design new antibacterial agents. The experimental work will be conducted on Neisseria meningitidis and Pseudomonas aeruginosa. N. meningitidis infection can lead to progression from bacteremia and meningitis to septic shock syndrome and death in a matter of hours. It often affects young children, with devastating effects (e.g. deafness, amputation). P. aeruginosa causes life-threatening infection in immunodepressed and cystic fibrosis patients. It is a pathogen that is found in hospitals and since the existing strains are resistant to all available antibiotics, it causes significant financial stress to healthcare systems worldwide. Any new ways of combating these bacteria, via targeting or exploiting their DSB pathways, could have immediate beneficial effects for affected groups or groups in danger and will alleviate part of the enormous financial burden (more than 1.2 billion pounds per year in the world) that bacterial infections cause.

This project also offers an ideal training opportunity for the PDRA who will be exposed to many aspects of biochemistry, immunology, cell biology and bioinformatics. The broad scope and diverse challenges offered by this project will train the PDRA who will develop a strong multidisciplinary attitude to biological science.

Beyond academic impact, there is a massive societal need for novel antibacterial strategies that will be designed with the problem of antibiotic resistance and pathogen adaptation in mind. The knowledge that will be acquired within this project has the potential to have a significant contribution towards the realisation of this goal and towards overcoming pathogenic bacteria.