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Abstract
Legionella pneumophila are facultative intracellular bacteria and accidental human pathogens that can cause a severe and deadly pneumonia known as Legionnaires disease. In order to establish and maintain an intracellular niche within host cells, Legionella utilize a type IVB secretion system to inject around 300 effector proteins into the eukaryotic host, to manipulate pathways and processes to the benefit of the bacterium. One such effector protein, LegC7, was originally identified as causing cell death when expressed in yeast, and was noted to disrupt vesicle trafficking. In this work we demonstrate that LegC7 specifically disrupts the localization of cargos that traffic through the endosomal system, while leaving alternative trafficking pathways unaltered. We also demonstrate that the toxicity of LegC7 is reduced or eliminated upon deletion of a subset of early endosome trafficking genes, centered on the early endosome Rab GTPase VPS21 and the early endosome tethering complex CORVET. Expression of LegC7 causes a mislocalization of Vps8p, the Rab binding subunit of the CORVET complex, and LegC7-RFP displays a high degree of colocalization with this subunit. Thus, disruption of the CORVET complex likely explains the specific endosomal disruption that occurs upon LegC7 expression. Disruption of the normal endosome maturation pathway likely prevents progression of the infected phagosome into the traditional lysosomal fusion pathway. Finally, we show that LegC7 is degraded upon deletion of the ER glycosylated protein chaperone EMP47, which was detected as interacting with LegC7 based on mass spectrometry. This evidence, along with the observation that LegC7-RFP is not mislocalized upon deletion of endosome trafficking genes, suggests the LegC7 disrupts endosome trafficking indirectly. Mutant versions of LegC7 show that endosomal disruption and toxicity are two distinct events, with the toxicity attributed to the interaction with Emp47p. A model of LegC7 acting in the ER fits with the high degree of manipulation that Legionella is known to exert on the ER to Golgi trafficking pathway during infection. This work suggests a more involved role of glycosylated proteins and the Emp47p chaperone in the proper functioning of the eukaryotic endosomal system.