Host Cell S Phase Restricts Legionella pneumophila Intracellular Replication by Destabilizing the Membrane-Bound Replication Compartment

Abstract

Legionella 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.

Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella ‐containing vacuole

“…The secondary screen took advantage of a fluorescence readout we previously described to detect disrupted ΔsdhA vacuoles in knockdown macrophages combined with shRNA knockdown in these primary cells (Creasey & Isberg, 2012). Terminally differentiated BMDMs were used to facilitate microscopic readout, and to avoid the complication of having a portion of the cells in S phase, which results in a high proportion of unstable vacuoles (de Jesus-Diaz, Murphy, Sol, Dorer, & Isberg, 2017). This strategy allowed subtle differences in vacuole integrity to be detected.…”

Legionella pneumophila translocated translation inhibitors are required for bacterial-induced host cell cycle arrest

“…We previously demonstrated that S phase provides a toxic environment for Legionella pneumophila growth and that S phase-infected cells do not progress through the cell cycle after L. pneumophila challenge (18). Therefore, avoidance of S has the potential to protect this pathogen from antimicrobial events.…”

“…There is also evidence supporting a role for pathogens in modulating tumor progression (17), although the role of such control in supporting disease is still unknown. Recently, studies performed in our laboratory determined that host cell cycle regulatory proteins control L. pneumophila growth (18). We demonstrated that the G 1 and G 2 /M phases of the host cell cycle are permissive for bacterial replication, while S phase provides a toxic environment for bacterial replication.…”

“…We demonstrated that the G 1 and G 2 /M phases of the host cell cycle are permissive for bacterial replication, while S phase provides a toxic environment for bacterial replication. L. pneumophila that attempts to initiate replication in S phase shows poor viability, due to a failure to control vacuole integrity that leads to cytosolic exposure of the bacterium and bacterial cell lysis resulting from cytoplasmic innate immune surveillance (11, 18)…”

“…Cell cycle progression plays an important role in the intracellular growth of L. pneumophil, as emphasized by specific S phase toxicity towards this pathogen (18). Therefore, bacterial control of the host cell cycle can limit exposure of the pathogen to antimicrobial events that are cycle-specific.…”