title: High-Content Imaging Reveals Expansion of the Endosomal Compartment duringCoxiella burnetiiParasitophorous Vacuole Maturation.
abstract: Coxiella burnetiiis an obligate intracellular pathogen and the causative agent of human Q fever. Replication of the bacterium within a large parasitophorous vacuole (PV) resembling a host phagolysosome is required for pathogenesis. PV biogenesis is a pathogen driven process that requires engagement of several host cell vesicular trafficking pathways to acquire vacuole components. The goal of this study was to determine if infection byC.burnetiimodulates endolysosomal flux to potentially benefit PV formation. HeLa cells, infected withC. burnetiior left uninfected, were incubated with fluorescent transferrin (Tf) for 0-30 min, and the amount of Tf internalized by cells quantitated by high-content imaging. At 3 and 5 days, but not 1 day post-infection, the maximal amounts of fluorescent Tf internalized by infected cells were significantly greater than uninfected cells. The rates of Tf uptake and recycling were the same for infected and uninfected cells; however, residual Tf persisted in EEA.1 positive compartments adjacent to large PV after 30 min of recycling in the absence of labeled Tf. On average,C. burnetii-infected cells contained significantly more CD63-positive endosomes than uninfected cells. In contrast, cells containing large vacuoles generated byChlamydia trachomatisexhibited increased rates of Tf internalization without increased CD63 expression. Our results suggest thatC. burnetiiinfection expands the endosomal system to increase capacity for endocytic material. Furthermore, this study demonstrates the power of high-content imaging for measurement of cellular responses to infection by intracellular pathogens.
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health Hamilton, MT, USA.
authors.forename: Charles L
authors.initials: CL
authors.lastname: Larson
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health Hamilton, MT, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Heinzen
mesh_terms: D002690:Chlamydia Infections / Q000382:microbiology; D002692:Chlamydia trachomatis / Q000166:cytology / Q000502:physiology; D016997:Coxiella burnetii / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism* / Q000472:pathogenicity; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism / Q000382:microbiology* / Q000648:ultrastructure*; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D008247:Lysosomes; D008856:Microscopy, Fluorescence; D010588:Phagosomes / Q000382:microbiology; D060149:Tetraspanin 30 / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism / Q000382:microbiology*
chemical_list: C554586:CD63 protein, human; D060149:Tetraspanin 30
keywords: Chlamydia trachomatis; Coxiella burnetii; endocytic trafficking; endosome; fluorescence microscopy; intracellular pathogen; lysosome