A bacterial c-di-GMP responsive enzyme modulates LPS structure and triggers immune evasion

Bacteria thrive in a multitude of environments and bacterial pathogens encounter stressful and harsh conditions when they colonize their host. In order to cope with nutrient limitations, aggression by the immune system and many other environmental fluctuations, most bacteria have evolved sophisticated regulatory networks. They can put in place specialized detectors by which they sense the conditions encounter in a specific niche and modulate their genetic program so that a specific set of genes are activated/repressed in order to reorganize the physiology of the cell so that it is optimally adapted.
In many bacteria the initial detection could be the responsibility of so called two component regulatory systems, which detect a stimulus and transfer this information to a regulator that will modulate gene expression through direct control of gene expression. In many cases the transmission of this information might not be direct but make use of an intracellular second messenger, which is a small molecule that will relay this information.
I the present proposal we will study one such messenger that is called c-di-GMP. We know that this molecule is central to the lifestyle of one of the most dreadful gram-negative bacterial pathogen, Pseudomonas aeruginosa, which not only emerges as a multi-resistant organisms but has always been extremely resistant even to standard anti-biotherapy due to a poorly permeable cell envelope and high efflux capacity thus retaining drugs outside. Furthermore P. aeruginosa can adopt very different infection strategies and lifestyles, and this can lead to acute infection or chronic infection. In the latter case the bacterium build a city of microbe that is known as biofilm, and this bacterial community becomes even more resistant to antibiotic treatment and eradication by the immune system.
Whereas c-di-GMP is a very simple molecule it is absolutely central to the lifestyle changes and adaptation and by elevating the intracellular levels of c-di-GMP bacteria turns immediately into the biofilm mode by slowing down motility, increasing the production of an extracellular matrix which glue all cells together, activating molecular weapon which will help fight against resident bacteria or the immune system. This is a lot of tasks for one single molecule but the beauty of the mechanisms is that it is many-fold. The c-di-GMP can be made or broken by a large number of enzymes (cyclase to make or phosphodiesterase to break) and can bind a large number of proteins, which will in turn modify their activity. For example, c-di-GMP can bind a regulator that is then activated and that will drive gene expression. It can bind some specific proteins, which will modify their conformation, which will then interact for example with the flagellar motor, thus stopping rotation and arresting movement to enter the biofilm mode.
In previous work we have identified an very novel c-di-GMP signalling pathway and found that the molecule which is synthesized by the cyclase SadC is directly transferred onto a protein that we called through a direct protein-protein interaction. We have shown that this protein, that we called WarA has an enzymatic activity, namely methyltransferase, and modifies the surface of the bacterium by changing the structure of the LPS. The LPS is well known in gram-negative bacteria to be one key element (also called Pathogen associated Motif Pattern or PAMP) that triggers the immune system. We have shown that a bacterial strain devoid of this enzyme WarA, becomes immediately recognized by the immune system, as we demonstrate the recruitment of immune cells on the infection site in the transparent zebra fish model.
This is a very exciting discovery and the present proposal is aiming at understanding the cascade of molecular events in a way that we will be able to manipulate this pathway to make P. aeruginosa readily detected by the immune system and more accessible to antibiotic treatment.

Bacteria sense stimuli in the environment which triggers regulatory networks which optimize adaptation to a specific niche. Second messengers can relay this information at the intracellular level and c-di-GMP is a major player in this game. It is particularly the case for our model organism Pseudomonas aeruginosa, which is a dreadful bacterial pathogen. Infections are associated with high morbidity and mortality, particularly severe in hospitals and with immunosuppressed or cystic fibrosis patients. The severity of the infections, the versatility of the organism and its high resistance to antimicrobials (P from ESKAPE) are urgently calling for novel therapeutic strategies. c-di-GMP signalling emerges as an appropriate target. Seminal work from my lab and others showed that it is central to a switch in lifestyles and between chronic or acute infections. At high c-di-GMP levels genes promoting biofilm formation are turned on, establishing a resilient community barely eliminated by the immune system. Major questions about c-di-GMP signalling is how it occurs and how specificity is established. One P. aeruginosa cell plays with c-di-GMP levels using 40 different enzymes, cyclases and phosphodiesterase, that make or break the molecule. Distinct signalling pathways result in specific output, e.g. motility, biofilm, AMR. We found using the cyclase SadC, that direct protein-protein interaction accounts for specificity. We showed that the cyclase transfers c-di-GMP directly on an enzyme we called WarA. c-di-GMP binding turns on WarA activity which modifies LPS composition/structure. Using zebra fish as a model, we showed that a warA mutant is less able to escape the immune system. This is an original finding and a new phenotypic output for c-di-GMP signalling. This work has just been accepted in Nature Microbiology and the present proposal aims at deciphering the molecular mechanisms involved and at exploiting our findings to proposed new therapeutic approaches.

The beneficiaries of this research are as follows:

1) The UK academic community with interests in molecular microbiology, bacterial communication, biofilm and infection. The insights gained will be of use to academic researchers interested in developing and applying the general principles of bacterial signalling to understanding how they contribute to the molecular basis of biofilm formation, human infections and immune evasion. Furthermore the characterization of new c-di-GMP binding proteins and new c-di-GMP binding motifs will be of benefit to evolutionary biologists by providing example of diversification.

2) Pharmaceutical industries and Biotech will also benefit from this research as it will potentially provide novel insights to develop new therapeutic approaches and new antimicrobial or vaccine strategies against dreadful bacterial infections which can turn into development of antimicrobial resistant populations. The c-di-GMP dependent signalling system is conserved within a number of human Gram-negative pathogens and the mechanistic information produced in the research could be applicable to other human pathogens in addition to P. aeruginosa. The identification of novel c-di-GMP binding proteins can be seen as potential novel targets and can thus obviously result in the development of new drugs. The potential development and manufacture of novel anti-infective strategies by European (Sanofi-Aventis for whom I act as a consultant) and UK-based Pharma or Biotech based on c-di-GMP signalling will be of direct benefit to the European and UK economy as such therapeutics if successful would have a world-wide market.

3) Public sector health care professionals will benefit from the research in terms of an improved knowledge about the cause of P. aeruginosa infections and possible new treatment plans. P. aeruginosa is the 3rd most commonly-isolated nosocomial pathogen accounting for 10% of hospital-acquired infections, with 10,000 cases each year in UK. The development of novel therapeutic approaches would improve quality of life and health in the UK, especially in the context of chronic infections with resilient bacterial biofilms established once the bacteria have escaped elimination by the immune system. Specialist healthcare workers treating cystic fibrosis patients would particularly benefit from the work as in late stage CF, the sole microorganism left in CF patient lungs is P. aeruginosa, which is firmly and chronically established and will lead to the patient death.

4) The UK knowledge-based economy will benefit from the interdisciplinary training of the PDRA working on the research project and who is effectively acting as a research co-investigator. Such trained RA (and associated PhD, masters and undergraduate students) is likely to benefit the biotechnology and pharmaceutical industries, as well as the academic base in the UK. We therefore anticipate medium term economic benefits arising from a well-trained UK and international research base, reflected in maintaining internationally competitive research.