Date Published: December 21, 2016
Publisher: Public Library of Science
Author(s): Eleanor A. Latomanski, Patrice Newton, Chen Ai Khoo, Hayley J. Newton, Tomoko Kubori.
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.
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. Transmission to humans occurs via inhalation of contaminated aerosols. Human infection can lead to an acute, pneumonia-like illness, or proceed to a chronic disease state in which endocarditis can manifest . During natural infection, 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. After internalization, Coxiella passively traffics through the endolysosomal pathway [2, 3]. The developing vacuole obtains markers typical of early and late endosomes, such as EEA1 and Rab7, and finally matures to a lysosome [4, 5]. Here, 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 . The active form of Coxiella is the replicative large cell variant (LCV), distinct from the environmentally stable small cell variant (SCV) .
The identification and characterization of Dot/Icm effector proteins in Coxiella is an active field of research that has been significantly bolstered by the recent advances in axenic culture and genetic manipulation of Coxiella. However, ascribing functions to these unique effectors remains challenging. Here, we have identified a host factor and pathway, namely FCHO2 and clathrin-mediated endocytosis, that are targeted by the effector protein Cig57. Clathrin-mediated endocytosis plays an essential role in all nucleated cells. Uptake and recycling of a variety of molecules, from plasma membrane receptors to iron is dependent upon the clathrin pathway. Additionally, clathrin is responsible for the trafficking of early endosomal vesicles to and from the trans-Golgi network playing an important role in delivering cargo proteins to their destination organelles [38, 39].