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. 2006 Sep 4;203(9):2177-89.
doi: 10.1084/jem.20060766. Epub 2006 Aug 28.

NF-kappaB translocation prevents host cell death after low-dose challenge by Legionella pneumophila

Vicki P Losick  1 , Ralph R Isberg
Affiliations

NF-kappaB translocation prevents host cell death after low-dose challenge by Legionella pneumophila

Vicki P Losick et al. J Exp Med. .

Abstract

Legionella pneumophila, the causative agent of Legionnaires' disease, grows within macrophages and manipulates target cell signaling. Formation of a Legionella-containing replication vacuole requires the function of the bacterial type IV secretion system (Dot/Icm), which transfers protein substrates into the host cell cytoplasm. A global microarray analysis was used to examine the response of human macrophage-like U937 cells to low-dose infections with L. pneumophila. The most striking change in expression was the Dot/Icm-dependent up-regulation of antiapoptotic genes positively controlled by the transcriptional regulator nuclear factor kappaB (NF-kappaB). Consistent with this finding, L. pneumophila triggered nuclear localization of NF-kappaB in human and mouse macrophages in a Dot/Icm-dependent manner. The mechanism of activation at low-dose infections involved a signaling pathway that occurred independently of the Toll-like receptor adaptor MyD88 and the cytoplasmic sensor Nod1. In contrast, high multiplicity of infection conditions caused a host cell response that masked the unique Dot/Icm-dependent activation of NF-kappaB. Inhibition of NF-kappaB translocation into the nucleus resulted in premature host cell death and termination of bacterial replication. In the absence of one antiapoptotic protein, plasminogen activator inhibitor-2, host cell death increased in response to L. pneumophila infection, indicating that induction of antiapoptotic genes is critical for host cell survival.

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Figures

Figure 1.
Figure 1.
Use of flow cytometry to isolate U937 cells associated with L. pneumophila. L. pneumophila strains Dot+-GFP and dotA −-GFP were introduced onto U937 macrophage monolayers at MOI = 1, allowed to incubate for 1 or 8 h, and infected cells were isolated by fluorescence sorting (Materials and methods). (A) Schematic diagram of sorting experiment. (B) One bacterial division occurs during the 8-h incubation. For each sorted GFP+ fraction, the cells were plated on coverslips, and the number of bacteria per phagosome was determined by fluorescence microscopy. (C and D) Examples of the fluorescence profile of sorted, uninfected macrophages (solid gray line); sorted, infected macrophages at 1 hai (gray dashed line); or sorted, infected macrophages at 8 hai (black dashed line) for Dot+-GFP (C) or dotA −-GFP (D), respectively. The percentage of max (% of max) indicates the number of cells relative to the peak fraction of cells.
Figure 2.
Figure 2.
Microarray analysis of infected cells shows induction of host cell genes in response to functional Dot/Icm secretion system. Pearson hierarchical cluster analysis of genes differentially expressed relative to uninfected, unsorted reference cells. 799 genes are displayed. Shown are mock (uninfected cells, sorted/reference), dotA −-GFP (MOI = 10; unsorted dotA −-GFP/reference), dotA −-GFP (MOI = 1; sorted dotA −- GFP/reference), dimB −-GFP (sorted dimB −-GFP/reference), or Dot+-GFP (sorted Dot+-GFP/reference) at 1 and 8 hai. The same reference preparation of uninfected U937 cells was used for each comparison. Each condition is represented as the mean from three independent microarrays, each of which was performed on preparations of RNA from different infections. Genes were judged to be induced or repressed if they were twofold up- or down-regulated in comparison to unsorted, uninfected reference cells (P < 0.05 as determined by the t test). Green represents repressed genes (<1), black represents equally expressed genes (reference 1), and red represents induced genes (>1) on a log scale of 0.1–10. The complete list of differentially expressed genes from the hierarchical cluster is shown in Table S1. Microarray data has been deposited in the NCBI Gene Expression Omnibus under accession no. GSE5551.
Figure 3.
Figure 3.
Induction of host genes after contact with Dot+-GFP predicted to be up-regulated. qPCR analysis was performed on infected U937 cells sorted 8 hai. Mock (sorted, uninfected); sorted, Dot+-GFP–infected; and sorted, dotA −-GFP–infected cells are shown. Relative gene expression represents the normalized value of the denoted sorted cDNA versus unsorted, uninfected cDNA.
Figure 4.
Figure 4.
NF-κB p65 translocation is dependent on functional type IV secretion system. (A) U937 macrophages (top) or A/J BM macrophages (bottom) were incubated with Dot+ or dotA − at MOI = 1 for 6 h. NF-κB p65 localization and L. pneumophila (Lp) were visualized by immunofluorescence microscopy after probing with anti–L. pneumophila (red) or anti-p65 (green). Arrowheads point to bacteria, and arrows point to macrophages with associated bacteria. Bar, 10 μm. Time course of NF-κB p65 translocation in infected U937 cells (B and D) or A/J BM macrophages (C and E). Each graph shows the percentage of cells with nuclear p65 staining. (B and C) The following incubations were analyzed: uninfected cells, cells harboring Dot+, cells harboring dotA −, and cells harboring dotA − introduced at MOI = 10. (D and E) The following incubations were analyzed: uninfected cells, cells harboring Dot+ pMMB207, cells harboring dotA − pMMB207, cells harboring icmS − pMMB207, and cells harboring icmS −/pMMB207icmS +. Means + SE from three coverslips from a representative experiment are displayed. A total of 100 infected cells were counted per coverslip.
Figure 5.
Figure 5.
NF-κB activation is required for host cell survival after L. pneumophila infection. A/J BM macrophages were transduced with retroviruses expressing either GFP or the dominant negative IκBDN-IRES-GFP construct (reference 38). Transduced A/J macrophages were then incubated with the Dot+ or dotA − strains at MOI = 1, and NF-κB p65 translocation and bacteria were visualized by immunofluorescence microscopy at 2 and 6 hai. (A) Images are examples of transduced macrophages harboring bacteria at 6 hai. NF-κB p65 and L. pnenumophila (Lp) were stained with same flour. Bacteria are marked with arrowheads, and cell nuclei are marked with arrows. Nuclear morphology was observed by Hoechst DNA staining. Bar, 10 μm. (B) NF-κB p65 translocation and (C) cell death (condensed nuclei) were observed by immunofluorescence microscopy and quantitated. Means + SE from three independent experiments are explained. A total of 100 cells harboring bacteria were counted per experiment.
Figure 6.
Figure 6.
NF-κB translocation is necessary for host cell survival and bacterial replication. A/J BM macrophages were pretreated for 2 h in the presence or absence of CAPE at 10 μg/ml (reference 39). Cells were incubated with the Dot+ or dotA − strains at MOI = 1, and NF-κB p65 translocation and bacteria were visualized by immunofluorescence microscopy at 1 and 6 hai. Nuclear morphology was observed by Hoechst DNA staining. (A) Examples of p65 staining and nuclear morphology at 6 hai. Bar, 10 μm. (B) NF-κB p65 translocation and cell death (condensed nuclei) were quantitated at 1 or 6 hai. (C) Percentage of condensed nuclei at 15 hai. (D) Bacterial replication at 15 hai. The percentage of cells having phagosomes bearing the denoted number of bacteria is shown. The only cells counted were those harboring bacteria. Means + SE of three coverslips from a representative experiment are given. U, uninfected cells; −, untreated cells; +, cells treated with CAPE.
Figure 7.
Figure 7.
PAI-2–dependent and MyD88/Nod1-independent effects associated with L. pneumophila control of NF-κB translocation. (A) BM macrophages derived from C57BL/6 or C57BL/6 pai-2 −/− mice were incubated with L. pneumophila Dot+-flaA − at MOI = 1 for the indicated times. Nuclear morphology was observed by Hoechst DNA staining, and the percentage of infected cells with condensed nuclei was quantitated. **, P < 0.0005 as determined by the Student's t test. (B) BM macrophages from C57BL/6 and C57BL/6 myd88 −/− mice were incubated with the Dot+, Dot+-flaA −, or dotA − strains at MOI = 1 or 5 for the noted times. The percentage of infected cells with NF-κB p65 staining in the nucleus was quantitated by immunofluorescence microscopy. (C) BM macrophages from C57BL/6 nod1 +/− heterozygous or C57BL/6 nod1 −/− homozygous mice were incubated with the Dot+, Dot+-flaA −, or dotA − strains at MOI = 1 for the noted times, and nuclear p65 was determined as in B. Means + SE from three independent experiments are shown.
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References

    1. Fraser, D.W., T.R. Tsai, W. Orenstein, W.E. Parkin, H.J. Beecham, R.G. Sharrar, J. Harris, G.F. Mallison, S.M. Martin, J.E. McDade, et al. 1977. Legionnaires' disease: description of an epidemic of pneumonia. N. Engl. J. Med. 297:1189–1197. - PubMed
    1. Fields, B.S. 1996. The molecular ecology of Legionellae. Trends Microbiol. 4:286–290. - PubMed
    1. Horwitz, M.A. 1983. Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes. J. Exp. Med. 158:1319–1331. - PMC - PubMed
    1. Horwitz, M.A. 1983. The Legionnaires' disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J. Exp. Med. 158:2108–2126. - PMC - PubMed
    1. Tilney, L.G., O.S. Harb, P.S. Connelly, C.G. Robinson, and C.R. Roy. 2001. How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane. J. Cell Sci. 114:4637–4650. - PubMed

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