Abstract
Legionella pneumophila translocates multiple bacterial effector proteins into host cells to direct formation of a replication vacuole for the bacterium. The emerging consensus is that formation of this compartment involves recruitment of membrane material that traffics between the endoplasmic reticulum (ER) and Golgi. To investigate this model, a targeted approach was used to knock down expression of proteins involved in membrane trafficking, using RNA interference in Drosophila cells. Surprisingly, few single knockdowns of ER–Golgi transport proteins decreased L. pneumophila replication. By analyzing double-stranded RNAs in pairs, combinations were identified that together caused defects in intracellular replication, consistent with the model that membrane traffic funnels into the replication vacuole from multiple sources. In particular, simultaneous depletion of the intermediate compartment and Golgi-tethering factor transport protein particle together with the ER SNARE protein Sec22 reduced replication efficiency, indicating that introduction of lesions at distinct sites in the secretory system reduces replication efficiency. In contrast to knockdowns in secretory traffic, which required multiple simultaneous hits, knockdown of single cytosolic components of ER-associated degradation, including Cdc48/p97 and associated cofactors, was sufficient to inhibit intracellular replication. The requirement for the Cdc48/p97 complex was conserved in mammalian cells, in which replication vacuoles showed intense recruitment of ubiquitinated proteins, the preferred substrates of Cdc48/p97. This complex promoted dislocation of both ubiquitinated proteins and bacterial effectors from the replication vacuole, consistent with the model that maintenance of high-level replication requires surveillance of the vacuole surface. This work demonstrates that L. pneumophila has the ability to gain access to multiple sites in the secretory system and provides the first evidence for a role of the Cdc48/p97 complex in promoting intracellular replication of pathogens and maintenance of replication vacuoles.
Highlights
The agent of Legionnaires pneumonia, the Gram negative Legionella pneumophila, creates a replication vacuole that is dependent on the function of the Dot/Icm protein translocation machine, a member of the specialized type IV conjugative secretion machinery family [1,2]
After Drosophila Kc167 cells were treated with double-stranded RNA (dsRNA) for 3 d, the cells were incubated with L. pneumophila–green fluorescent protein (GFP) for 48 h, fixed, and screened visually for increased or decreased intracellular replication based on quantification of vacuole size
That other endoplasmic reticulum (ER)–Golgi transport targets did not effect intracellular replication was surprising, as we found that Golgi morphology was disrupted by treatment of several of the dsRNAs directed against proteins involved in export of vesicles from the ER
Summary
The agent of Legionnaires pneumonia, the Gram negative Legionella pneumophila, creates a replication vacuole that is dependent on the function of the Dot/Icm protein translocation machine, a member of the specialized type IV conjugative secretion machinery family [1,2]. This system transports at least 30 different protein substrates across target membranes [3,4,5,6,7]. Adding to the complexity of the LCV is that it Editor: Daniel Portnoy, University of California Berkeley, United States of America
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