Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis

Abstract

Successful macrophage colonization by Coxiella burnetii, the cause of human Q fever, requires pathogen-directed biogenesis of a large, growth-permissive parasitophorous vacuole (PV) with phagolysosomal characteristics. The vesicular trafficking pathways co-opted by C. burnetii for PV development are poorly defined; however, it is predicted that effector proteins delivered to the cytosol by a defective in organelle trafficking/intracellular multiplication (Dot/ Icm) type 4B secretion system are required for membrane recruitment. Here, we describe involvement of clathrin-mediated vesicular trafficking in PV generation and the engagement of this pathway by the C. burnetii type 4B secretion system substrate Coxiella vacuolar protein A (CvpA). CvpA contains multiple dileucine [DERQ]XXXL [LI] and tyrosine (YXXΦ)-based endocytic sorting motifs like those recognized by the clathrin adaptor protein (AP) complexes AP1, AP2, and AP3. A C. burnetii ΔcvpA mutant exhibited significant defects in replication and PV development, confirming the importance of CvpA in infection. Ectopically expressed mCherry-CvpA localized to tubular and vesicular domains of pericentrosomal recycling endosomes positive for Rab11 and transferrin receptor, and CvpA membrane interactions were lost upon mutation of endocytic sorting motifs. Consistent with CvpA engagement of the endocytic recycling system, ectopic expression reduced uptake of transferrin. In pull-down assays, peptides containing CvpA-sorting motifs and full-length CvpA interacted with AP2 subunits and clathrin heavy chain. Furthermore, depletion of AP2 or clathrin by siRNA treatment significantly inhibited C. burnetii replication. Thus, our results reveal the importance of clathrincoated vesicle trafficking in C. burnetii infection and define a role for CvpA in subverting these transport mechanisms.type IV secretion | vesicular fusion

Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development

“…One of the key features of CCVs is their remarkable fusogenicity, which allows these compartments to intercept and merge with the endocytic, autophagy, secretory, and recycling pathways (28)(29)(30). To date, however, little is known about the mechanisms responsible for CCV biogenesis as well as the protein and lipid composition of CCVs.…”

Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)

“…CLTC (clathrin heavy chain) is also enriched on the CCV membrane [17,18]. Clathrin-mediated endocytosis is the process by which host cells internalize extracellular material, termed cargo, from the plasma membrane.…”

“…Cargo molecules are recognized by adaptor protein complexes, such as AP2 (adaptor related protein complex 2), which form a link to CLTC itself, which acts as a scaffold forming a cage-like structure around clathrin-coated vesicles [19]. The Coxiella Dot/Icm effector CvpA has been shown to bind AP2, and is required for both intracellular replication of Coxiella and CLTC recruitment to the CCV [17]. Gene silencing studies demonstrate that CLTC and AP2 are important for CCV biogenesis and intracellular replication of Coxiella .…”

“…Gene silencing studies demonstrate that CLTC and AP2 are important for CCV biogenesis and intracellular replication of Coxiella . In the absence of CLTC, CCVs remain small and support minimal Coxiella replication [17,18]. More recently, the Coxiella effector Cig57 was shown to interact with FCHO2 (FCH domain only 2), an accessory protein in clathrin-mediated endocytosis, and contribute to Coxiella virulence, as Cig57 is essential for intracellular replication of Coxiella and CCV biogenesis [13,18].…”

Identification of Novel Coxiella burnetii Icm/Dot Effectors and Genetic Analysis of Their Involvement in Modulating a Mitogen-Activated Protein Kinase Pathway

“…Three effectors (AnkG, CaeA, and CaeB) were shown to inhibit apoptosis in macrophages (26,27), and an additional effector, CvpA, was found to subvert clathrin-mediated vesicular trafficking (28). Important clues for possible additional functions that might be performed by Icm/Dot effectors came from several studies that investigated the host pathways and factors required for C. burnetii intracellular growth.…”