Identification of novel drugs targeting Francisella tularensis secreted effectors – TulareDrug

A bioinformatic approach will be performed to identify candidate effectors.
Secretion into the host cell will be assessed thanks to two reporter fusions namely b-lactamase and adenlylate cyclase
The role of the identified effector in the virulence will be characterized both in vitro and in vivo in a mouse model of tularemia using both the surrogate strain Francisella novicida and the highly pathogenic strain Francisella tularensis.
The human host target will be identified by yeast two hybrid.
A drug screening assay will be performed in order to find lead compounds disrupting the interaction.

We have made ??mutants, identified secreted proteins and identified putative partners by yeast two hybrid screen.

The validation of the interactions is currently under way as is the characterization of the mutants.

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Francisella tularensis is a facultative intracellular bacterium that causes tularaemia. F. tularensis is one of the most infectious bacterial pathogen for man. Indeed, in cases of pneumonic tularemia, the lethal dose for humans is under 10 bacteria. This high infectiosity has led to the classification of F. tularensis as a class A bioterrorism agent.

The ability of F. tularensis to cause disease depends on its ability to infect macrophages, escape from the phagosome and replicate within the host cytosol. The molecular mechanisms enabling this infectious cycle are largely unknown. A genetic locus, the Francisella pathogenicity island, has been shown to be required for escape into the cytosol and intracellular survival and is thought to encode an atypical type VI secretion system. The current hypothesis in the field is that F. tularensis secretes effector proteins into the host cytosol using the FPI-encoded secretion apparatus and possibly other secretion machinery to hijack host cell machinery likely through direct bindings to host proteins.

Here we propose to i) identify novel F. tularensis effector proteins secreted into the host cell and required for virulence ii) identify their eukaryotic binding partners by yeast two hybrid technology and decipher their molecular functions; iii) screen two chemical libraries to identify drugs disrupting the interaction of a key F. tularensis effector protein with its eukaryotic binding partner and leading to blockage of the infectious cycle.

To achieve these goals, we have assembled a unique Consortium of complementary skills, combining two research laboratories with expertise in bacterial genetics and host cell biology, a clinical laboratory associated with the national reference center for tularemia with expertise with highly virulent and clinical strains of F. tularensis and a dynamic French SME possessing the most advanced applied technological resources to uncover and characterize protein-protein interactions and screen drugs to inhibit these interactions.

This project should lead to the characterization of novel virulence mechanisms and to the identification of novel antibiotic molecules specifically disrupting an interaction between a F. tularensis effector protein and its host binding partner leading to blockage of the intracellular infectious cycle of this highly virulent bacterium.