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.

Many organisms respond to DNA damage by inducing expression of DNA repair genes. We find that the human stomach pathogen Helicobacter pylori instead induces transcription and translation of natural competence genes, thus increasing transformation frequency. Transcription of a lysozyme-like protein that promotes DNA donation from intact cells is also induced. Exogenous DNA modulates the DNA damage response, as both recA and the ability to take up DNA are required for full induction of the response. This feedback loop is active during stomach colonization, indicating a role in the pathogenesis of the bacterium. As patients can be infected with multiple genetically distinct clones of H. pylori, DNA damage induced genetic exchange may facilitate spread of antibiotic resistance and selection of fitter variants through re-assortment of preexisting alleles in this important human pathogen.

Interactions between hosts and pathogens are complex, so understanding the events that govern these interactions requires the analysis of molecular mechanisms operating in both organisms. Many pathogens use multiple strategies to target a single event in the disease process, confounding the identification of the important determinants of virulence. We developed a genetic screening strategy called insertional Mutagenesis And Depletion (iMAD) that combines bacterial mutagenesis and RNA interference, to systematically dissect the interplay between a pathogen and its host. We used this technique to resolve the network of proteins secreted by the bacterium Legionella pneumophila to promote intracellular growth, a critical determinant of pathogenicity of this organism. This strategy is broadly applicable, allowing the dissection of any interface between two organisms involving numerous interactions.

SummaryHelicobacter pylori is a genetically diverse bacterial species, owing in part to its natural competence for DNA uptake that facilitates recombination between strains. Inter-strain DNA recombination occurs during human infection and the H. pylori genome is in linkage equilibrium worldwide. Despite this high propensity for DNA exchange, little is known about the factors that limit the extent of recombination during natural transformation. Here, we identify restrictionmodification (R-M) systems as a barrier to transformation with homeologous DNA and find that R-M systems and several components of the recombination machinery control integration length. Type II R-M systems, the nuclease nucT and resolvase ruvC reduced integration length whereas the helicase recG increased it. In addition, we characterized a new factor that promotes natural transformation in H. pylori, dprB. Although free recombination has been widely observed in H. pylori, our study suggests that this bacterium uses multiple systems to limit inter-strain recombination.