Abstract
Coxiella burnetii, the etiologic agent of Q fever, is a Gram-negative obligate intracellular bacterium. It has been previously described that both the endocytic and autophagic pathways contribute to the Coxiella replicative vacuole (CRV) generation. Galectins are β-galactoside-binding lectins that accumulate in the cytosol before being secreted via a non-conventional secretory pathway. It has been shown that Galectin-3, -8, -9 monitor bacteria vacuolar rupture and endosomal and lysosomal loss of membrane integrity through binding of host glycans exposed in the cytoplasm after membrane damage. Using microinjection of fluorescence-coupled dextrans, a FRET assay, and galectins distribution, we demonstrate that Coxiella infection actually result in transient phagosomal/CRV membrane damage in a Dot/Icm-dependent manner. We also show the association of different adaptor molecules involved in autophagy and of LC3 to the limiting membrane of the CRV. Moreover, we show that upon autophagy inhibition, the proportion of CRVs labeled with galectins and less acidified increases which is associated with bacteria replication impairment. Based on these observations, we propose that autophagy can facilitate resealing of intracellular damaged membranes.
Highlights
The obligate intracellular bacterium Coxiella burnetii, the etiologic agent of Q-fever (Voth and Heinzen, 2007), is a highly infectious human pathogen responsible for a global zoonotic disease called Q fever
It has been previously demonstrated that C. burnetii transits the endocytic and autophagic pathways generating an acidic and replicative niche with phagolysosomal characteristics which develops in a large Coxiella replicative vacuole” (CRV) at 48 h of infection (Akporiaye et al, 1983; Maurin et al, 1992; Heinzen et al, 1999; Grieshaber et al, 2002)
To demonstrate the significance of the membrane damaged caused by Coxiella and the role of autophagy in this process, mouse embryonic fibroblast (MEF) wt and Atg5−/− cells were infected with C. burnetii and following 48 h of infection, the cells were fixed, subjected to immunofluorescence against Gal3 and analyzed by confocal microscopy
Summary
The obligate intracellular bacterium Coxiella burnetii, the etiologic agent of Q-fever (Voth and Heinzen, 2007), is a highly infectious human pathogen responsible for a global zoonotic disease called Q fever. C. burnetii inhabits mainly monocytes/macrophages but it can infect a wide variety of host cells in vitro (Voth and Heinzen, 2007) It directs the biogenesis of a large membrane-bound compartment called “Coxiella replicative vacuole” (CRV). Galectins belong to a family of proteins highly conserved through evolution involved in several biological events They are able to recognize specific carbohydrates in complex macromolecules located on cell membranes or in the extracellular matrix (ECM) through a carbohydrate recognition domain (CRD), which interacts with the structure (Gal β1 → 4 GlcNAc). Our results indicate that adaptor molecules involved in autophagyrelated signaling pathways associate to the limiting membrane of the CRV, likely to control distinct host cell responses upon pathogen infection. We propose that the autophagic pathway favors Coxiella infection by contributing to the repair of the transiently damaged replicative compartment
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