Linking c-di-GMP signalling and the Gac/Rsm signal transduction pathway

Pseudomonas aeruginosa is a gram-negative bacterium that thrives in a multitude of environmental niches including soil, moist surfaces or plants. It is also an opportunistic human pathogen causing infections associated with high levels of morbidity and mortality. Epidemiological studies have shown that no significant differences in the virulence potential of clinical or environmental isolates could be observed, which suggests that the available arsenal of virulence factors might also be useful to fight eukaryotic predators that may be encountered in the environment (e.g. protozoans and metazoans). As such, P. aeruginosa is a versatile organism and its adaptive potential to fluctuating environmental cues relies on numerous and complex signalling pathways. These pathways determine whether P. aeruginosa adopts a motile/toxic or sessile/biofilm lifestyle. These lifestyles directly relate to acute or chronic infection mode.
The decision making process for choosing in between lifestyles is governed by major regulatory pathways which are antagonistic but also cross-talk via positive and negative feedback loops. These involve Quorum Sensing, or the second messenger molecule c-AMP, which are both master regulators for promoting production of bacterial virulence factors such as toxins. In contrast the so-called, Gac/Rsm pathway, which involves molecular sensors for probing environmental conditions, and the second messenger molecule c-di-GMP are main players to control the formation of biofilm.
A biofilm is a bacterial population attached on a surface and held together by a sticky extracellular matrix made of polysaccharide, DNA or adhesive proteins. Bacteria embedded in such matrix form a very stable population which in the environment can resist various stresses such as the current of a river flow when attached on rocks, UV irradiation, dehydration or nutrient starvation for example. For pathogens, once the bacteria establish as a biofilm in one particular tissue or organ, such as in the lungs of cystic fibrosis patients in case of P. aeruginosa, it is basically impossible to eradicate. The population became highly resistant to attack by the immune system or to antibiotic therapy. This way a chronic and persistent infection establishes and morbidity reaches a very high level.
In our project we will investigate the intimate connections that may exist in between the formation/dispersion of a P. aeruginosa biofilm and the two regulatory pathways Gac/Rsm on the one hand and c-di-GMP on the other hand. We want to build on our expertise and pioneering work in the characterization of the Gac/Rsm pathway and on our recent demonstration that this route intersects with the c-di-GMP signaling pathway. We want to establish the molecular links between these two main players and understand how it may then act on the formation of the biofilm and beyond this how it exerts an antagonistic action on the production of virulence factors such as toxins. Indeed, these regulatory pathways are at the intersection of a Doctor Jekyll and Mister Hyde game, in which bacteria hesitate to balance either towards an aggressive and toxic lifestyle or towards a more silent and insidious lifestyle under the form of a biofilm.
In conclusion, the regulatory network we will investigate is not unique to the human pathogen P. aeruginosa but common to most gram-negative bacteria for c-di-GMP and to most Pseudomonas species for Gac/Rsm including plant growth promoters (Pseudomonas putida and Pseudomonas fluorescens) or plant pathogens (Pseudomonas syringae). We believe that a precise molecular understanding of this circuitry will allow to develop small molecules designed for manipulating the fate of bacteria, subverting and influencing their decision making process to our own benefit. This study will have strong implications in several areas including, Ecology and Agriculture (niche/root colonization), Medical and Therapeutic approaches and Fundamental and Basic Sciences.

Microorganisms developed strategies to adapt to changes in their surrounding using regulatory cascades. Pseudomonas aeruginosa is a versatile bacterium with high adaptive potential, which is found in most environments and also a nosocomial pathogen.
P. aeruginosa thrives as a motile organism, i.e. planktonic lifestyle, or establish a biofilm, i.e. sessile lifestyle. Biofilm is associated with persistent infection while planktonic lifestyle correlates with systemic dissemination. We contributed the characterization of a network, which is a key switch in P. aeruginosa lifestyles. It involves two sensors, LadS and RetS, which antagonistically influence the Gac/Rsm pathway. Gac/Rsm controls level of small RNAs, which sequester the repressor RsmA. As a consequence biofilm formation is promoted and motility is reduced. RsmA sequestration also causes a switch in proteins secretion systems with an increase in T6SS, whereas the cytotoxic activity of the T3SS is shut down.
In recent years we added complexity by showing that i) another regulatory pathway centred on the phosphorelay HptB runs parallel to RetS/LadS but intersects with Gac/Rsm and ii) that c-di-GMP, a universal signalling molecule for biofilm formation, is connected to Gac/Rsm.
We aim at understanding the molecular connections existing between the signal transduction pathways involving RetS and HptB and c-di-GMP signalling. Signalling cascades involving c-di-GMP are poorly understood and c-di-GMP binding proteins are of different types. It is predicted that the transfer of c-di-GMP from the biosynthetic enzymes to cognate effectors may involve a cascade of protein-protein interactions. We will use genetic and biochemical approaches to elucidate the link between Gac/Rsm and c-di-GMP, which will be our case study. We shall also bring further light on c-di-GMP signalling in general by using a bacterial two-hybrid library of P. aeruginosa and probing protein-protein interactions with c-di-GMP metabolic enzymes.

Pseudomonas aeruginosa is a major cause of hospital-acquired infection, with an estimated 10,000 cases each year in UK. Infection is severe and life-threatening, leading to pneumonia or septicaemia. P. aeruginosa is also dreaded by cystic fibrosis patients. Up to 80% carry the bacteria in their lungs and infection is proving fatal.
Regulation of P. aeruginosa gene expression in relation with biofilm formation and chronic infection is an intense field of research involving top class laboratories all over the world. Together with Prof Stephen Lory's laboratory (Harvard Medical School), my laboratory has been a major player in this area. Our discovery of bacterial sensors that switch on and off genes involved in virulence and biofilm formation, in an antagonistic manner, has been considered as a seminal finding in this area of research and is of major interest for the academic community as well as for clinicians and pharmaceutical companies. The sensors we discovered, LadS and RetS, influence the activity of the Gac/Rsm signalling pathway, which has been described in several other organisms. Our more recent work demonstrates that the universal c-di-GMP second messenger is tightly connected to the Gac/Rsm pathway to signal and decide on the bacterial behaviour. Although our research is basic research, molecular understanding of infection strategy employed by such an important human pathogen may lead to biomedical applications, new antimicrobials and societal impact. Furthermore the existence of a similar pathway in the plant pathogen Pseudomonas syringae will not restrict applications to human pathogens and may thus have impact in agriculture as well. Finally the pathway is crucial in typically environmental organisms such as Pseudomonas putida or Pseudomonas fluorescens and certainly influences their ability to colonize their environmental niches which suggest that understanding this regulatory network will have broader impact on understanding the fate of microbial population in nature and thus on ecology.
Based on the visibility of my laboratory in the field and a broad network of collaborators, it will be another achievement to organize a small size (50 participants) meeting on the site of Imperial College (near the end of the project). The topic will be "Regulation of gene expression in P. aeruginosa, biofilm and chronic infection". It will attract the main UK players in this field (Nottingham University, Cambridge University, Southampton University) together with other groups from France (CNRS Marseille, Institut Pasteur) and US (Harvard Medical School, University of Washington, Binghamton University). This way we will be in control of disseminating our findings not only by attending regular conferences and by publishing in high impact factor journals. Pharmaceutical companies representative (Sanofi-Aventis), and journal editors (Nature) will also be invited to attend the conference. Alain Filloux has a good expertise in conference organization, having been the organizer of the Pseudomonas meeting (Marseille 2005) and chair of the Gordon conference on Microbial adhesion and cell signalling (Newport 2009).