Tooth diseases: Porphyromonas active secretion of toxin effectors – TOOTHPASTE

Periodontitis and gum diseases are considered as major public health concerns as roughly 36% of the world population (20% in industrialized countries) suffer of serious tooth tissue damages and tooth decay. The main microorganism responsible for periodontitis and gingivitis is the oral pathogenic bacterium Porphyromonas gingivalis. Infection by this Gram-negative bacterium causes severe lesions in periodontal tissues such as gingiva or the alveolar bone by disrupting the tooth-supporting structure. These damages are induced by a cocktail of specialized toxin proteins secreted by Porphyromonas, called gingipains. Gingipains act as adhesins or proteases that help the bacterium to adhere to periodontal tissues and to promote gingival tissue invasion by the degradation of matrix proteins, fibrinogen and collagen. The active release of gingipains at the bacterial cell surface is catalyzed by a recently identified protein complex called the Type IX secretion system (T9SS). This secretion system is composed of 10-14 subunits – encoded by the por genes – that are thought to assemble a trans-envelope channel that specifically recruits the gingipains and transport them to the cell surface, where they are processed to be active (cleavage of a C-terminal sequence and glycosylation). The mechanism by which the T9SS selects the gingipains is not understood but all gingipains carry a conserved C-terminal sequence, called CTD, which may serve as secretion signal, suggesting that the secretion of gingipains relies on a mechanism common to all these enzymes.
While the risks of gum diseases are diminished with preventive care, no efficient treatment is currently available to fight and eradicate Porphyromonas or to interfere with the activities of the gingipains. P. gingivalis being a strictly anaerobe pathogen, medical treatments consist to inject hydrogen peroxide in the gum or the tooth tissues. However, this approach has a very limited impact. Treatments are currently developed to target the gingipains. Here again, the gingipains is a broad family of enzymes, carrying very diverse activities. A molecule that interferes with the activity of one of these enzymes would have no or moderate consequence on the others. The most obvious solution is to identify molecules that will interfere with a mechanism shared by all gingipains such as those that (i) specifically block the expression of the T9SS genes, (ii) specifically inhibit the function of the T9SS and/or (iii) specifically target the gingipain secretion signal and interfere with its recruitment to the secretion apparatus. There is therefore a need to gain insights into the composition, assembly and mode of action of the T9SS. The present proposal wishes to tackle these questions. First, the different Por subunits will be characterized in details by in vivo localization, topology and protein-protein interaction approaches and by biophysical methods (light scattering, gel filtration, interactions by surface plasmon resonance or biolayer interferometry). Then, tri-dimensional structures of these proteins or domains will be determined when possible by X-ray diffraction or electron microscopy and further experiments targeting T9SS sub-complexes will aim to define the general architecture of the T9SS core complex. Finally, we propose to better understand the initial stages of gingipain recruitment. The two teams involved in this project are internationally recognized for the study of bacterial secretion systems and of large macromolecular complexes and have complementary expertise (molecular biology, biochemistry, biophysical and structural biology).