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. 2007 Nov;189(22):8224-32.
doi: 10.1128/JB.00898-07. Epub 2007 Sep 14.

Flagella Facilitate Escape of Salmonella From Oncotic Macrophages

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Free PMC article

Flagella Facilitate Escape of Salmonella From Oncotic Macrophages

Gen-ichiro Sano et al. J Bacteriol. .
Free PMC article

Abstract

The intracellular parasite Salmonella enterica serovar Typhimurium causes a typhoid-like systemic disease in mice. Whereas the survival of Salmonella in phagocytes is well understood, little has been documented about the exit of intracellular Salmonella from host cells. Here we report that in a population of infected macrophages Salmonella induces "oncosis," an irreversible progression to eukaryotic cell death characterized by swelling of the entire cell body. Oncotic macrophages (OnMphis) are terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling negative and lack actin filaments (F-actin). The plasma membrane of OnMphis filled with bacilli remains impermeable, and intracellular Salmonella bacilli move vigorously using flagella. Eventually, intracellular Salmonella bacilli intermittently exit host cells in a flagellum-dependent manner. These results suggest that induction of macrophage oncosis and intracellular accumulation of flagellated bacilli constitute a strategy whereby Salmonella escapes from host macrophages.

Figures

FIG. 1.
FIG. 1.
OnMφ formation. (A to C) OnMφs (arrowheads) formed in cultures of RAW 264.7 (A) and J774A.1 (B) cells and peritoneal macrophages (C) 4 h after infection at an MOI of 100 with wild-type Salmonella strain χ3306 opsonized with anti-Salmonella antisera. Scale bars = 20 μm. Also see Movie S1 in the supplemental material. (D) Percentage of OnMφs (dotted and filled bars) based on the total number of macrophages. The filled bars indicate the fraction of OnMφs with vigorously moving intracellular Salmonella bacilli observed with the light microscope. The data are means ± standard errors of the means. (E) Effects of opsonization and MOI on OnMφ formation. RAW 264.7 cells were infected with wild-type Salmonella strain χ3306 opsonized with anti-Salmonella antisera (AS) or normal mouse sera (N) or not opsonized (−) at MOIs of 5 and 100. (F) Effects of IFN-γ on OnMφ formation. RAW 264.7 cells were preincubated with 0, 1, 10, and 100 nM IFN-γ for 24 h and then infected with wild-type Salmonella strain χ3306. Infected cells were analyzed 4 h after infection. (G to J) Transmission electron micrographs of uninfected RAW 264.7 cells (G) and infected RAW 264.7 cells containing intracellular Salmonella bacilli (H to J). Scale bars = 2 μm. Salmonella bacilli are located in discrete SCVs (H), in a large common SCV (I), or in the diluted cytosol (J).
FIG. 2.
FIG. 2.
F-actin dissociation in OnMφs. (A) Time-lapse video microscopy of RAW 264.7 cells forming an OnMφ 4 h after infection. The white dots indicate the cell boundary. (B to D) Confocal microscopy of uninfected (left panels) and infected (right panels) RAW 264.7 cells 4 h after infection. OnMφs are indicated by arrowheads. Samples were stained for F-actin (red) (B to D), Salmonella (green) (B to D), and CD18 (blue) (B), β-tubulin (blue) (C), or vimentin (blue) (D). (E) Confocal laser microscopy analysis of infected RAW 264.7 cells. Four hours after wild-type Salmonella strain χ3306 infection, infected cells were fixed and stained for TUNEL (pink), Salmonella (green), and F-actin (red). The arrowheads indicate TUNEL-negative, Salmonella-positive, and F-actin-negative macrophages (oncotic). The arrows indicate TUNEL-positive macrophages (apoptotic). There were few TUNEL-negative and F-actin-negative cells lacking intracellular Salmonella bacilli (asterisks). Scale bars = 20 μm.
FIG. 3.
FIG. 3.
Biochemical features of Salmonella-induced OnMφs. (A) Giemsa staining of OnMφs containing more than 50 bacilli (arrowhead) 4 h after infection of RAW 264.7 cells. (B) Calcium levels in RAW 264.7 cells. The [Ca2+]i of uninfected macrophages (n = 25), OnMφs containing motile Salmonella (n = 21), and H2O2-treated apoptotic macrophages (n = 12) were determined. Asterisk, P < 0.0001 for a comparison with uninfected cells. (C) Caspase-1 and caspase-3 activities in OnMφs. Uninfected, Salmonella-infected, and H2O2-treated apoptotic macrophages (control) were examined. OnMφs containing motile Salmonella bacilli are indicated by arrowheads. (D) TUNEL-positive macrophages (left panel, red nuclei, arrows) containing a few GFP-expressing Salmonella bacilli (green) and a TUNEL-negative OnMφ (right panel, arrowhead) containing numerous bacilli (green) 4 h after infection. Note that the images in the left and right panels are from the same field and are the same magnification. DAPI staining is blue. (E) Permeability of the plasma membrane of OnMφs (arrowheads). Live macrophages with intact membranes are stained green. Dead macrophages with permeable membranes are stained red (arrows). BF, bright field; DIC, differential interference contrast; IF, immunofluorescence. Scale bars = 20 μm.
FIG. 4.
FIG. 4.
Motility of Salmonella bacilli in OnMφs is driven by flagella. (A) Confocal microscopy of OnMφs (arrowheads) and apoptotic macrophages (arrow) derived from RAW 264.7 cells. Four hours after infection, macrophages were fixed and stained for F-actin (red) and flagella (green). Apoptotic cells are TUNEL positive (pink). (B) OnMφ formation by flagellum-deficient Salmonella. RAW 264.7 cells were infected with the χ3181-derived mutant lacking flagella (ΔfliA mutant). The bars indicate the percentages of OnMφs; vigorously moving intracellular Salmonella bacilli were not observed. The data are means ± standard errors of the means. AS, anti-Salmonella antisera; N, normal mouse sera. (C) Traces of Salmonella (lines) were recorded and analyzed for the wild-type strain (wt) and the ΔfliA mutant by time-lapse microscopy in a liquid medium (left panels) and in OnMφs 4 h after infection (right panels). The white dots indicate cell boundaries. Each trace was analyzed for 5 s. Scale bars = 20 μm. (D) Maximal velocity (Vmax) of wild-type and ΔfliA Salmonella strains in liquid medium and in OnMφs. The data are means ± standard deviations from five traces shown in panel C. Two asterisks, P < 0.001 for a comparison with wild-type controls.
FIG. 5.
FIG. 5.
Escape of Salmonella bacilli from host cells. (A) Time-lapse microscopy showing Salmonella bacilli (arrowhead) exiting from a swollen macrophage at 5 h postinfection. The white dots indicate the host cell boundary. (B) Numbers of Salmonella bacteria exiting from a single host cell in panel A per minute for 70 min. (C) Histogram showing the percentages of the total macrophages containing different numbers of flagellated Salmonella bacilli per cell 6 h after infection. Cross-hatched bars, macrophages with intact F-actin; filled bars, macrophages with F-actin dissociation. (D) Confocal microscopy of RAW 264.7 cells infected with the wild-type Salmonella strain (wt) or the mutant lacking flagella (ΔfliA). Two hours after infection, infected RAW 264.7 cells were transferred to gentamicin-free medium, cultured for an additional 6 h, fixed, and stained for F-actin (red), Salmonella (green), and DAPI (blue). Scale bar = 20 μm.
FIG. 6.
FIG. 6.
Model for escape of Salmonella from OnMφs. Salmonella bacilli are confined to SCVs after they are engulfed by macrophages. Some infected cells undergo apoptosis, pyroptosis, or autophagy. Other infected cells become OnMφs (undergo oncosis), contain flagellated motile Salmonella bacilli, and lack F-actin, but they contain intact DNA. Intracellular Salmonella bacilli continuously exit from the cell. N, host cell nucleus.

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