RNA Interference Analysis of Legionella in Drosophila Cells: Exploitation of Early Secretory Apparatus Dynamics

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. Legionella pneumophila is a pathogenic bacterium that causes Legionnaires pneumonia. Immune cells, called macrophages, engulf the bacterium and attempt to kill it. Legionella avoids this killing and instead grows inside the macrophage, creating a growth niche using host cell components. The bacterium directs the formation of its replication niche by injecting bacterial proteins, called effectors, into the host cell. These effectors hijack host functions. In this study, the authors identify some of the host pathways that the bacterium hijacks. The authors used macrophage-like cells derived from fruit flies because protein function can be disrupted in these cells using a technique called RNA interference, which destroys the RNA messages that encode for proteins, resulting in directed loss of these proteins. Candidate proteins were chosen to disrupt based upon previous knowledge about the biology of Legionella. This report highlights two observations that contribute to our understanding of the biology of Legionella. Surprisingly, the absence of some host components could be tolerated because other host components could take their place. One exception to this rule was a protein complex on the outside of the Legionella replication vacuole that may help the bacterium deliver its proteins to appropriate sites in the host cell.