Abstract
Coxiella burnetii is an intracellular bacterial pathogen that infects alveolar macrophages and replicates within a unique lysosome-derived vacuole. When Coxiella is trafficked to a host cell lysosome the essential Dot/Icm type IV secretion system is activated allowing over 130 bacterial effector proteins to be translocated into the host cytosol. This cohort of effectors is believed to manipulate host cell functions to facilitate Coxiella-containing vacuole (CCV) biogenesis and bacterial replication. Transposon mutagenesis has demonstrated that the Dot/Icm effector Cig57 is required for CCV development and intracellular replication of Coxiella. Here, we demonstrate a role for Cig57 in subverting clathrin-mediated traffic through its interaction with FCHO2, an accessory protein of clathrin coated pits. A yeast two-hybrid screen identified FCHO2 as a binding partner of Cig57 and this interaction was confirmed during infection using immunoprecipitation experiments. The interaction between Cig57 and FCHO2 is dependent on one of three endocytic sorting motif encoded by Cig57. Importantly, complementation analysis demonstrated that this endocytic sorting motif is required for full function of Cig57. Consistent with the intracellular growth defect in cig57-disrupted Coxiella, siRNA gene silencing of FCHO2 or clathrin (CLTC) inhibits Coxiella growth and CCV biogenesis. Clathrin is recruited to the replicative CCV in a manner that is dependent on the interaction between Cig57 and FCHO2. Creation of an FCHO2 knockout cell line confirmed the importance of this protein for CCV expansion, intracellular replication of Coxiella and clathrin recruitment to the CCV. Collectively, these results reveal Cig57 to be a significant virulence factor that co-opts clathrin-mediated trafficking, via interaction with FCHO2, to facilitate the biogenesis of the fusogenic Coxiella replicative vacuole and enable intracellular success of this human pathogen.
Highlights
The intracellular bacterial pathogen Coxiella burnetii is the causative agent of human Q fever, a zoonotic disease with the potential to cause life-threatening complications
We show FER/CIP homology only protein 2 (FCHO2) is required for optimal Coxiella-containing vacuole (CCV) formation, and establish that Cig57, interacting with FCHO2 via a tyrosine-based endocytic sorting motif, subverts clathrin to the CCV to facilitate normal vacuole biogenesis and intracellular replication of Coxiella
Cig57 alone did not activate reporter gene expression, as Saccharomyces cerevisiae (Y2H Gold, Clontech) carrying pGBKT7-cig57 could not grow on quadruple dropout (QDO) yeast minimal media (YMM) plates (-Trp, -Leu, -His, -Ade)
Summary
The intracellular bacterial pathogen Coxiella burnetii is the causative agent of human Q fever, a zoonotic disease with the potential to cause life-threatening complications. Human infection can lead to an acute, pneumonia-like illness, or proceed to a chronic disease state in which endocarditis can manifest [1]. Coxiella predominantly invades alveolar macrophages, and in order to replicate intracellularly, a spacious and fusogenic lysosome-derived vacuole, termed the Coxiella-containing vacuole (CCV), is established by the pathogen. With an internal pH of approximately 4.8, and in the presence of proteolytic and degradative enzymes, Coxiella becomes metabolically active and will direct the expansion of the CCV before replicating to large numbers [6]. The active form of Coxiella is the replicative large cell variant (LCV), distinct from the environmentally stable small cell variant (SCV) [7]
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