The Coxiella burnetii Dot/Icm System Delivers a Unique Repertoire of Type IV Effectors into Host Cells and Is Required for Intracellular Replication

Abstract

Coxiella burnetii, the causative agent of human Q fever, is an intracellular pathogen that replicates in an acidified vacuole derived from the host lysosomal network. This pathogen encodes a Dot/Icm type IV secretion system that delivers bacterial proteins called effectors to the host cytosol. To identify new effector proteins, the functionally analogous Legionella pneumophila Dot/Icm system was used in a genetic screen to identify fragments of C. burnetii genomic DNA that when fused to an adenylate cyclase reporter were capable of directing Dot/Icm-dependent translocation of the fusion protein into mammalian host cells. This screen identified Dot/Icm effectors that were proteins unique to C. burnetii, having no overall sequence homology with L. pneumophila Dot/Icm effectors. A comparison of C. burnetii genome sequences from different isolates revealed diversity in the size and distribution of the genes encoding many of these effectors. Studies examining the localization and function of effectors in eukaryotic cells provided evidence that several of these proteins have an affinity for specific host organelles and can disrupt cellular functions. The identification of a transposon insertion mutation that disrupts the dot/icm locus was used to validate that this apparatus was essential for translocation of effectors. Importantly, this C. burnetii Dot/Icm-deficient mutant was found to be defective for intracellular replication. Thus, these data indicate that C. burnetii encodes a unique subset of bacterial effector proteins translocated into host cells by the Dot/Icm apparatus, and that the cumulative activities exerted by these effectors enables C. burnetii to successfully establish a niche inside mammalian cells that supports intracellular replication. Coxiella burnetii is a Gram-negative intracellular bacterium that can cause the human disease Q fever. A type IV secretion system in C. burnetii called Dot/Icm is functionally similar to the Dot/Icm system of Legionella pneumophila. Here we used L. pneumophila to screen a C. burnetii library for genes encoding effector proteins. We identified 18 effectors that are unique to C. burnetii and show that when they are expressed in eukaryotic cells they localize to specific compartments and can mediate changes in host cell physiology. Comparative genomic analysis revealed plasticity among these novel effector proteins that could be related to the different manifestations of disease exhibited by these clinical isolates of C. burnetii. A transposon insertion mutation in the dot/icm locus revealed for the first time that type IV secretion is essential for C. burnetii replication inside mammalian host cells and that the delivery of effectors requires Dot/Icm function. Thus, this study conclusively shows that the C. burnetii Dot/Icm system is an essential determinant for intracellular replication, and identifies a repertoire of unique effector proteins with novel functions delivered by this system that could be important for disease phenotypes.