Final Report Summary - Q-SCREEN (Large-scale identification of Coxiella burnetii virulence factors)
In the last decade, cellular microbiology has stressed the importance of studying pathogens in relation to their host. However, the effective, global identification of bacterial virulence determinants and their characterization require the development of new, automated approaches. With this project, we have set up protocols for the multi-phenotypic screen of bacterial factors that are involved in host cell invasion and colonization. Our approach integrates transposon mutagenesis, genomics, bioinformatics and fluorescence-based functional assays that have been adapted for the large-scale identification of virulence factors from virtually any intracellular bacterium. The advantage of our screening technique lies in the possibility of analyzing every bacterial mutations for multiple phenotypes, such as 1) internalization within the host/cell; 2) intracellular replication and 3) cytotoxicity, simultaneously. The emerging bacterial pathogen Coxiella burnetii is an excellent model system to apply our strategy. To date, very little is known about the bacterial factors that regulate Coxiella interactions with their host and previous in silico identification of putative Coxiella virulence factors provides an excellent database to cross-reference bioinformatics analysis with our functional assays. With our proof-of-concept screen we have generated and investigated over 1000 Coxiella mutations and identified several bacterial proteins involved in the key steps of host cells colonization. Among these, we have identified the product of the Coxiella gene CBU_1260 as the first Coxiella invasin. Mutations of CBU_1260 severely affected Coxiella internalization and replication within host cells. Predictive analysis on the primary sequence of CBU_1260 revealed the presence of an OmpA-like domain and 4 unstructured loops exposed at the bacterial surface. Membrane fractionation experiments showed that Coxiella OmpA is indeed enriched in the outer membrane of Coxiella and by replacing each extracellular loop with a myc tag, we showed that loop 1 is essential for internalization. Importantly, perturbing the interactions between OmpA and a yet unidentified receptor at the surface of host cells effectively blocked Coxiella internalization. Finally, we have developed non-mammalian animal models to validate the role of Coxiella virulence determinants in vivo. Using Galleria mellonella larvae, we effectively reproduced the OmpA mutant phenotypes observed in cultured cells, demonstrating an essential role of the Coxiella invasin in developing a systemic infection. In summary, multi-phenotypic screening of host/pathogen interactions is an efficient method for the study of infectious diseases. We have applied this method to Coxiella infections and identified a bacterial protein that is essential for Coxiella internalization within non-phagocytic cells. Understanding how intracellular bacteria adhere to and invade their host is essential to 1) understand the cell biology of infection and identify the candidate targets of anti-infectious molecules and 2) to develop targeted vaccines. Of note, bacterial OmpA proteins are considered as new pathogen-associated molecular patterns (PAMPs) and are among the most immunodominant antigens in the outer membrane of Gram-negative bacteria. We are currently investigating the possibility of using OmpA to develop the first synthetic vaccine against Q fever (patent pending: EP 13005536.1).