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. 2019 Feb 19;116(8):3221-3228.
doi: 10.1073/pnas.1820093116. Epub 2019 Feb 4.

Legionella pneumophila Translocated Translation Inhibitors Are Required for Bacterial-Induced Host Cell Cycle Arrest

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Free PMC article

Legionella pneumophila Translocated Translation Inhibitors Are Required for Bacterial-Induced Host Cell Cycle Arrest

Asaf Sol et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The cell cycle machinery controls diverse cellular pathways and is tightly regulated. Misregulation of cell division plays a central role in the pathogenesis of many disease processes. Various microbial pathogens interfere with the cell cycle machinery to promote host cell colonization. Although cell cycle modulation is a common theme among pathogens, the role this interference plays in promoting diseases is unclear. Previously, we demonstrated that the G1 and G2/M phases of the host cell cycle are permissive for Legionella pneumophila replication, whereas S phase provides a toxic environment for bacterial replication. In this study, we show that L. pneumophila avoids host S phase by blocking host DNA synthesis and preventing cell cycle progression into S phase. Cell cycle arrest upon Legionella contact is dependent on the Icm/Dot secretion system. In particular, we found that cell cycle arrest is dependent on the intact enzymatic activity of translocated substrates that inhibits host translation. Moreover, we show that, early in infection, the presence of these translation inhibitors is crucial to induce the degradation of the master regulator cyclin D1. Our results demonstrate that the bacterial effectors that inhibit translation are associated with preventing entry of host cells into a phase associated with restriction of L. pneumophila Furthermore, control of cyclin D1 may be a common strategy used by intracellular pathogens to manipulate the host cell cycle and promote bacterial replication.

Keywords: Legionella pneumophila; cell cycle; innate immunity; intracellular growth; translation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
L. pneumophila Icm/Dot-dependent arrest of the host cell cycle is independent of cell cycle phase. (A and B) HeLa cells were synchronized by the double thymidine block method and challenged with WT or dotA3 L. pneumophila-GFP. At various times after uptake, cells were collected and analyzed by flow cytometry to determine cell cycle profile (Materials and Methods): 11.5 h (A) and 6.5 h (B) after release. (Left) Images with black filled distributions display cell cycle profiles at the time of contact with bacteria. “hpi” refers to the total time of contact with bacteria. Uninfected cells are indicated by black lines and infected cells by green lines.
Fig. 2.
Fig. 2.
L. pneumophila translocated protein synthesis inhibitors are required for the induction of host cell cycle arrest. (A) L. pneumophila blockade of DNA synthesis requires translocated protein synthesis inhibitors. RAW 264.7 macrophages were challenged with L. pneumophila WT, T4SS or ∆5 mutant for 1 h and then incubated with 20 µM EdU for an additional 2 h. At 3 hpi, cells were harvested and analyzed for DNA synthesis by flow cytometry (Materials and Methods). (B) Fraction of proliferating cells was quantified by using the same experimental conditions as A. (C) L. pneumophila protein synthesis inhibitors block amoebal proliferation. D. discoideum cells were incubated with 10 μM eFluor cell proliferation dye. Following staining, cells were challenged with WT, dotA3, or L. pneumophila-GFP∆5 for 24 h. At the noted time points, the amount of dye within cells was measured by flow cytometry. “DMSO” indicates fluorescence of cells with no dye; T0, fluorescence at time of bacterial challenge. (D) L. pneumophila expressing a single Lgt is sufficient to block host cell proliferation. RAW 264.7 macrophages were challenged with L. pneumophila WT or ∆5 complemented with pJB908 vector, Lgt3, or catalytic mutant of Lgt3 (Lgt3*). Following infection, proliferating cells were measured by EdU incorporation and analyzed by flow cytometry. Statistical analyses were performed by unpaired t test (**P < 0.01 and ***P < 0.001).
Fig. 3.
Fig. 3.
Legionella translocated protein synthesis inhibitors induce the rapid degradation of cyclin D1 in infected macrophages. (A) Lack of transcriptional effect on cyclin expression. Quantitative PCR analysis of Ccnd1 and Ccne1 transcripts in RAW 264.7 macrophages following L. pneumophila challenge for 2 h. Results are normalized based on Gapdh expression and presented as relative to uninfected cells. (B) RAW 264.7 macrophages were challenged with L. pneumophila WT, dotA3, or ∆5 for 2 h, and transcript levels of Ccnd1, Il6, and Egr1 were measured by quantitative PCR. Results are presented as relative to uninfected population. (CE) Reduced steady-state levels of cyclin D1 in response to L. pneumophila requires protein synthesis inhibition. RAW 264.7 macrophages were infected with WT, dotA3, or ∆5 L. pneumophila expressing GFP for 2 h and sorted based on fluorescence by flow cytometry. Following infection, the levels of cyclin D1 (C and D) and cyclin E1 (E) in infected (GFP+) and bystander (GFP) populations were measured by immunoblot. Graph in D indicates fold change of cyclin D1 in GFP+ compared with GFP cells. (F) RAW 264.7 macrophages were challenged with L. pneumophila WT or ∆5 in the absence or presence of 50 μg/mL CHX for 1 h and then incubated with 20 µM EdU for an additional 2 h. At 3 hpi, cells were harvested and analyzed for DNA synthesis by flow cytometry. (G) RAW 264.7 macrophages were infected with L. pneumophila WT or ∆5 as in F, and levels of cyclin D1 in GFP+ population were measured by immunoblot. Statistical analyses were performed by unpaired t test (*P < 0.05, **P < 0.01, and ***P < 0.001).
Fig. 4.
Fig. 4.
Ectopic expression of the LGTs is sufficient to block host translation and proliferation. (A and B) Transfected Lgts block translation. HEK 293T cells were transfected with GFP, Lgt1, Lgt3, or Lgt3* (catalytically dead) for 24 h. At 3 h before harvest, cells were methionine-starved for 1 h and incubated with 50 μg/mL CHX as appropriate. Cells were then incubated with AHA for 2 h, and de novo protein synthesis was measured by orthogonal chemistry and flow cytometry. (C) Lgts are sufficient to block proliferation. HEK 293T cells were transfected as in A. At 2 h before harvest, cells were incubated with 20 µM EdU, and de novo DNA synthesis was measured as in B. (D) Lowered steady-state levels of cyclin D1 in the presence of Lgts. HEK 293T cells were transfected as in A, and cyclin D1 levels were measured by immunoblot. (E and F) Overproduced cyclin D1 does not overcome Lgt inhibition. Cotransfection of Lgt3 or Lgt3* inactive enzyme with pcDNA3 harboring HA-cyclin D1 protein. At 24 h posttransfection, the levels of remaining cyclin D1 were measured by immunoblot (E). (F) The percentages of proliferating cells were measured by EdU incorporation as in C. Statistical analyses were performed by unpaired t test (*P < 0.05, **P < 0.01, and ***P < 0.001).
Fig. 5.
Fig. 5.
FZR1 silencing partially restores entry into S phase in response to L. pneumophila. (A) Challenge with L. pneumophila destabilizes cyclin D1. Densitometry analysis of cyclin D1 in uninfected RAW 264.7 macrophages and cells challenged with L. pneumophila in the presence of 10 μg/mL CHX. (B) Proteasome inhibition stabilizes cyclin D1. Immunoblot analysis of cyclin D1 in RAW 264.7 macrophages pretreated for 60 min with 10 μM of the proteasome inhibitor MG132, challenged with WT L. pneumophila for 2 h, and sorted by flow cytometry. (C) Schematic representation of the APC/C association with its coactivators in different phases of the cell cycle (Left). (Right) Efficiency of targeting Fzr1 with siRNA based on quantitative RT-PCR. (D) Depletion of FZR1 partially rescues proliferation of L. pneumophila-targeted cells. RAW 264.7 macrophages treated with siRNA against FZR1or nontargeting siRNA (NT) were challenged with L. pneumophila-GFP for 1 h, incubated with 20 µM EdU for an additional 2 h, and analyzed for de novo DNA synthesis by flow cytometry. (Top) Macrophages in absence of bacteria. Horizontal lines are used to represent gates that separate bystander from infected cells (based on lack of GFP fluorescence), and vertical lines are used to show percentage of S-phase cells based on the scans. (Bottom) Macrophages after 3 h total of infection based on gates determined above. Infected cells are those found in the GFP+ gate.

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References

    1. Isberg RR, O’Connor TJ, Heidtman M. The Legionella pneumophila replication vacuole: Making a cosy niche inside host cells. Nat Rev Microbiol. 2009;7:13–24. - PMC - PubMed
    1. Horwitz MA, Maxfield FR. Legionella pneumophila inhibits acidification of its phagosome in human monocytes. J Cell Biol. 1984;99:1936–1943. - PMC - PubMed
    1. Berger KH, Isberg RR. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol. 1993;7:7–19. - PubMed
    1. Segal G, Feldman M, Zusman T. The Icm/Dot type-IV secretion systems of Legionella pneumophila and Coxiella burnetii. FEMS Microbiol Rev. 2005;29:65–81. - PubMed
    1. Burstein D, et al. Genomic analysis of 38 Legionella species identifies large and diverse effector repertoires. Nat Genet. 2016;48:167–175. - PMC - PubMed

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