Abstract
ABSTRACTLegionella pneumophila grows within cells ranging from environmental amoebae to human macrophages. In spite of this conserved strategy of pathogenesis, identification of host factors that restrict L. pneumophila intracellular replication has not been extended outside components of the mammalian innate immune response. We performed a double-stranded RNA (dsRNA) screen against more than 50% of the Drosophila melanogaster annotated open reading frames (ORFs) to identify host cell factors that restrict L. pneumophila. The majority of analyzed dsRNAs that stimulated L. pneumophila intracellular replication were directed against host proteins involved in protein synthesis or cell cycle control. Consistent with disruption of the cell cycle stimulating intracellular replication, proteins involved in translation initiation also resulted in G1 arrest. Stimulation of replication was dependent on the stage of cell cycle arrest, as dsRNAs causing arrest during S phase had an inhibitory effect on intracellular replication. The inhibitory effects of S phase arrest could be recapitulated in a human cell line, indicating that cell cycle control of L. pneumophila replication is evolutionarily conserved. Synchronized HeLa cell populations in S phase and challenged with L. pneumophila failed to progress through the cell cycle and were depressed for supporting intracellular replication. Poor bacterial replication in S phase was associated with loss of the vacuole membrane barrier, resulting in exposure of bacteria to the cytosol and their eventual degradation. These results are consistent with the model that S phase is inhibitory for L. pneumophila intracellular survival as a consequence of failure to maintain the integrity of the membrane surrounding intracellular bacteria.
Highlights
Legionella pneumophila grows within cells ranging from environmental amoebae to human macrophages
Our data demonstrate that the G1 and G2/M phases of the host cell cycle are permissive for bacterial replication, while S phase is toxic for the bacterium
To identify host factors that modulate L. pneumophila intracellular replication, we developed a high-throughput, whole-genome double-stranded RNA (dsRNA) interference screen strategy
Summary
Legionella pneumophila grows within cells ranging from environmental amoebae to human macrophages. Translation inhibition occurs at a second level, as a result of a host response to L. pneumophila challenge In this case, degradation of the host mTOR protein in response to infection results in a block in translation initiation [20], and this strategy appears to be the primary mechanism of protein synthesis inhibition several hours after initial bacterial uptake [21]. Bacterial mutants that are defective for inhibiting translation elongation show no replication defect in mouse bone marrow-derived macrophages but have lowered intracellular growth in amoebae for unknown reasons [18], perhaps because the inhibitors block the host unfolded protein response [21, 22] a known form of innate immune protection in simple eukaryotes [23,24,25]. Inhibition of translation initiation, in particular, appears to bias mammalian cells toward producing proinflammatory cytokines, conceivably allowing the immune response to contain bacterial replication [20]
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