JB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Other Versions of this Article:
JB.00898-07v1
189/22/8224    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sano, G.-i.
Right arrow Articles by Matsuo, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sano, G.-i.
Right arrow Articles by Matsuo, K.
Journal of Bacteriology, November 2007, p. 8224-8232, Vol. 189, No. 22
0021-9193/07/$08.00+0     doi:10.1128/JB.00898-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Flagella Facilitate Escape of Salmonella from Oncotic Macrophages{triangledown} ,{dagger}

Gen-ichiro Sano,1 Yasunari Takada,1 Shinichi Goto,1 Kenta Maruyama,1 Yutaka Shindo,2 Kotaro Oka,2 Hidenori Matsui,3 and Koichi Matsuo1*

Department of Microbiology and Immunology, School of Medicine, Keio University, Tokyo 160-8582, Japan,1 Center for Biosciences and Informatics, School of Fundamental Science and Technology, Keio University, Kanagawa 223-8522, Japan,2 Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, Tokyo 108-8641, Japan3

Received 8 June 2007/ Accepted 31 August 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (OnM{phi}s) are terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling negative and lack actin filaments (F-actin). The plasma membrane of OnM{phi}s 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.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages detect and internalize bacterial pathogens in order to eliminate them from the host. Bacteria captured in phagosomes are usually killed by reactive oxygen species and nitrogen intermediates, acidification, starvation, lysosomal enzymes, antimicrobial peptides, or other activities. However, a subset of bacteria has evolved to survive within various types of vacuoles in macrophages.

The gram-negative bacterium Salmonella enterica serovar Typhimurium causes enterocolitis in humans and a systemic typhoid-like disease in mice. Intracellular Salmonella bacilli are found in Salmonella-containing vacuoles (SCVs), which localize around the endoplasmic reticulum (3, 18). Although SCVs are acidified, Salmonella bacilli can survive at low pH by remodeling their outer membrane (13). SCVs do not contain bactericidal enzymes such as NADPH oxidase, lysosomal enzymes, and inducible nitric oxide synthase (7, 57, 58). By contrast, Mycobacterium avium-containing vacuoles are not acidified, since the vacuolar proton ATPase is excluded from the membrane, and they do not mature into phagolysosomes (16). Legionella pneumophila-containing vacuoles are converted into rough endoplasmic reticulum-like structures and avoid recognition as phagosomes (30, 53). Little is known, however, about how any of these intracellular pathogens escapes from host phagocytes.

Genes in Salmonella pathogenicity island 1 (SPI1) are required mainly for host cell invasion and are down-regulated inside Salmonella-infected host cells (12). Within SCVs in macrophages, Salmonella also down-regulates synthesis of flagellar proteins in response to low pH (1, 12). Instead, intracellular survival and replication of Salmonella require genes located in a second locus, SPI2 (51), whose expression in host cells is activated by low osmolarity and acidic pH (11, 36).

While surviving in macrophages, Salmonella induces host cell death via diverse mechanisms at different times after infection (23, 31). Salmonella invasion protein B (SipB) is a major bacterial effector protein that induces macrophage cell death. SipB is encoded by SPI1 (24) and induces rapid (~1 h) macrophage cell death through activation of the host protein caspase-1, resulting in chromatin fragmentation and plasma membrane blebbing (5, 8, 29, 44). Although apoptosis and necrosis are terms that are widely used to define most forms of eukaryotic cell death, such terminology may be an oversimplification. Recently, a new concept has emerged, that apoptosis and necrosis are not mutually exclusive but rather necrosis is the end stage of any cell death, including apoptosis (15, 23, 31, 39). Salmonella induces cell death through caspase-3-dependent apoptosis (32), autophagy (via degradation of cytosolic components) (28), and pyroptosis, which is caspase-1 dependent but caspase-3 independent (4). Other host factors, such as c-Fos/AP-1, also affect Salmonella-induced macrophage cell death (40).

Here, we report that intracellular Salmonella triggers swelling of macrophages, an event termed oncosis (from "onkos," meaning swelling) (15, 39). We observed that "oncotic" macrophages (OnM{phi}s) are often packed with motile Salmonella bacilli and that later, flagellated Salmonella bacilli escape intermittently from OnM{phi}s, which then undergo necrotic cell death. These results reveal a novel strategy by which Salmonella survives in, accumulates in, and escapes from macrophages.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains and growth conditions. Bacterial strains were derived from the wild-type S. enterica serovar Typhimurium SR-11 strain {chi}3181 or {chi}3306 (Nalr) (25). Strain MS005 expressing green fluorescent protein was generated by introducing the pGFPmut3.1 plasmid (Clontech) into {chi}3306. Strain MS300 lacking flagella ({Delta}fliA) was constructed using bacteriophage P22 mutants (50) for transduction of fliA::Tn10 in the Salmonella LT2 strain KK2091 (35) into {chi}3181. Strain MS007 lacking phoP (phoP::aphT) has been described previously (41). Bacteria were grown in L broth or on L agar (Difco Laboratories) supplemented with appropriate antibiotics, including 15 µg/ml tetracycline, 100 µg/ml ampicillin (Sigma-Aldrich), and 25 µg/ml nalidixic acid (Nacalai Tesque).

Anti-Salmonella mouse serum. Female 6-week-old BALB/c mice (Charles River Japan) were orally inoculated with 1 x 108 CFU of an exponential-phase nonvirulent Salmonella {Delta}phoP strain in 20 µl phosphate-buffered saline (PBS) containing 0.01% (wt/vol) gelatin, as described previously (41). Mice were inoculated using three 10-day cycles, and sera were prepared 2 weeks later. Increases in the level of Salmonella lipopolysaccharide-specific immunoglobulin G (IgG) in serum (11.6 ± 8.2 µg/ml) were confirmed using an enzyme-linked immunosorbent assay (42). All experiments were performed according to institutional guidelines for animal experiments.

Cell culture and infection. RAW 264.7 (= ATCC TIB-71) and J774A.1 (= ATCC TIB-67) macrophages were seeded in Dulbecco modified Eagle medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal calf serum (Gibco) at a density of 3 x 105 cells/well in 24-well plates (Falcon) or on glass coverslips (Fischer Science). Peritoneal macrophages were prepared from female 6-week-old BALB/c mice as described previously (48). Salmonella strains were cultured at 37°C overnight without agitation in L broth supplemented with antibiotics. The following day, bacteria were diluted 1:30 in L broth containing antibiotics and agitated for about 2 h at 37°C until the optical density at 600 nm reached 0.3. Unless otherwise indicated, Salmonella strains were then opsonized by incubation with 0.01 volume of anti-Salmonella mouse serum for 15 min at 37°C. Macrophages were infected with opsonized bacteria at a multiplicity of infection (MOI) of 100 unless otherwise indicated. One hour later, cells were gently washed twice with PBS and once with medium and then immersed in complete medium supplemented with 10 µg/ml gentamicin (Gibco). Apoptotic RAW 264.7 cells were prepared by incubating cells in 0.1% H2O2-DMEM for 30 min at room temperature.

Transmission electron microscopy. Four hours after infection, RAW 264.7 cells were fixed for 1 h with 2.5% glutaraldehyde-PBS, washed three times in PBS, and postfixed in 1% OsO4 in Sorensen's buffer for 1 h. Samples were subsequently dehydrated in ethanol and flat embedded in epoxy resin (Agar100; Agar Scientific). Thin sections (60 to 80 nm) mounted on copper grids were stained with uranyl acetate and lead citrate and viewed at 120 kV with an electron microscope (JEM1230; JEOL).

Video analysis. Microscopy images (DMIL; Leica) were video recorded with a micro camera (GP-KS1000; Panasonic) using digital imaging software (Ulead VideoStudio version 6 and Adobe Premiere Pro 2.0). Bacterial velocity was calculated using an image-analyzing system (Move-tr/2D; Library).

Staining procedures. For immunostaining, DMEM was gently removed from cultures and cells were fixed for 5 min at room temperature in 4% paraformaldehyde-PBS. After two washes with PBS, cells were incubated in 0.1% Triton X-100-PBS for 5 min, washed twice with PBS, and stained. The primary antibodies, all diluted 1:100, were monoclonal anti-Salmonella goat antibody CSA-1 (Accurate Chemical & Scientific), anti-Salmonella flagellum H-i rabbit sera (Denka Seiken), and monoclonal anti-CD18 (M18/2) rat antibody (Pharmingen). The secondary antibodies, which were diluted 1:200, were Alexa Fluor 647-conjugated anti-rat IgG chicken antibody, Alexa Fluor 488-conjugated anti-rabbit IgG chicken antibody, and Alexa Fluor 647-conjugated anti-goat IgG rabbit antibody (Molecular Probes). F-actin was detected using Alexa Fluor 546 phalloidin (Molecular Probes) at a 1:100 dilution. Nuclei were stained with 4',6'-diamidino-2-phenylindole (DAPI) (Vector). A fluorescence microscope (Axiovert 135; Zeiss) and a confocal laser scanning microscope (FV1000; Olympus) were used for imaging. Giemsa staining was performed using Giemsa's solution (Nacalai Tesque). Caspase activities were detected by using APO LOGIX carboxyfluorescein caspase detection kits (Cell Technology) as described previously (2). The terminal deoxynucleotidyltransferase-mediated biotin-dUTP nick end labeling (TUNEL) assay was performed using a DeadEnd colorimetric TUNEL system kit (Promega). Plasma membrane integrity was analyzed using a LIVE/DEAD viability/cytotoxicity kit for mammalian cells (Molecular Probes).

Measurement of the intracellular Ca2+ concentration ([Ca2+]i). RAW 264.7 cells were infected with Salmonella in 35-mm glass bottom dishes (IWAKI) for 5 h. The culture medium was replaced with a solution containing 125 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 2 mM CaCl2, 1.2 mM KH2PO4, 6 mM glucose, 25 mM HEPES (pH 7.4), and the acetoxymethyl ester form of the radiometric fluorescent Ca2+ indicator fura-2 (fura-2 AM; Molecular Probes) at a concentration of 5 µM. Cells were incubated for 30 min, washed twice in the same buffer without fura-2 AM, and incubated in DMEM for 30 min. Fura-2 imaging was performed as described previously (20, 33).

Purification of CD11b-positive cells. Bacterial escape was analyzed after infected RAW 264.7 cells were separated from extracellular Salmonella bacilli by incubation with streptavidin-conjugated anti-mouse CD11b (M1/70; BD Biosciences) in DMEM supplemented with 10% fetal calf serum for 15 min at 37°C. Cells were purified using IMag streptavidin Particle Plus-DM and the IMag cell separation system (BD Biosciences). Bacterial escape was monitored in the absence of gentamicin.

Statistical analysis. Statistical comparisons were performed using Student's t test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Formation of OnM{phi}s after Salmonella infection. We first infected RAW 264.7 cells with wild-type Salmonella strain {chi}3306 at an MOI of 100 after bacteria were opsonized with anti-Salmonella antisera, and then we examined infected cells over time by light microscopy. From 4 to 6 h after infection, we observed that 10 to 20% of macrophages resembled inflated balloons and had flexible and translucent cell membranes characteristic of OnM{phi}s (15, 39). Moreover, bacteria were vigorously moving within 10 to 30% of the OnM{phi}s (Fig. 1A; see Movie S1 in the supplemental material). By contrast, in mock infection controls, we did not observe OnM{phi}s, indicating that Salmonella triggers OnM{phi} formation (data not shown). Formation of OnM{phi}s, as judged by swelling and the lack of a sharp boundary of the cell body, was also observed with J774A.1 and peritoneal macrophages (Fig. 1B, C, and D). A fraction of the OnM{phi}s in all three cell types contained motile Salmonella bacilli (Fig. 1D). Since vigorous movement of intracellular Salmonella bacilli was most readily observed with RAW 264.7 cells by light microscopy, we chose these cells for further analysis.


Figure 1
View larger version (50K):
[in this window]
[in a new window]

  		FIG. 1. OnM{phi} formation. (A to C) OnM{phi}s (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 {chi}3306 opsonized with anti-Salmonella antisera. Scale bars = 20 µm. Also see Movie S1 in the supplemental material. (D) Percentage of OnM{phi}s (dotted and filled bars) based on the total number of macrophages. The filled bars indicate the fraction of OnM{phi}s 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 OnM{phi} formation. RAW 264.7 cells were infected with wild-type Salmonella strain {chi}3306 opsonized with anti-Salmonella antisera (AS) or normal mouse sera (N) or not opsonized (–) at MOIs of 5 and 100. (F) Effects of IFN-{gamma} on OnM{phi} formation. RAW 264.7 cells were preincubated with 0, 1, 10, and 100 nM IFN-{gamma} for 24 h and then infected with wild-type Salmonella strain {chi}3306. 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).

 
We quantified OnM{phi} formation with and without opsonization and at MOIs of 5 and 100. Infection at the higher MOI, as well as opsonization, enhanced OnM{phi} formation, although even without opsonization and at an MOI of 5 OnM{phi}s consistently formed (Fig. 1E). We also asked whether pretreatment of RAW 264.7 cells with gamma interferon (IFN-{gamma}) enhanced OnM{phi} formation. IFN-{gamma} pretreatment followed by mock infection did not induce OnM{phi} formation (data not shown). By contrast, IFN-{gamma} pretreatment of RAW 264.7 cells for 24 h increased the proportion of OnM{phi}s (Fig. 1F) even though the number of OnM{phi}s containing motile Salmonella bacilli was not increased by IFN-{gamma} pretreatment (Fig. 1F). Since formation of OnM{phi}s containing motile Salmonella bacilli was most frequently observed without IFN-{gamma} pretreatment, with opsonization, and at an MOI of 100, we used these conditions in experiments described below. Using transmission electron microscopy, we next observed control uninfected RAW 264.7 cells (Fig. 1G) and infected RAW 264.7 cells containing abundant intracellular Salmonella bacilli (Fig. 1H to J). Salmonella bacilli were located either in discrete SCVs (Fig. 1H), in a large common SCV (Fig. 1I), or in the diluted cytosol, which was indicative of necrotic cell death (Fig. 1J).

Cytoskeletal reorganization. We next monitored OnM{phi} formation by time-lapse video microscopy 4 to 6 h after infection. Prior to swelling, Salmonella movement was limited to a small region of the cytoplasm. Swelling was often complete in 5 min (Fig. 2A). Such morphological changes enabling formation of gigantic SCVs suggest that there is cytoskeletal reorganization (59).


Figure 2
View larger version (51K):
[in this window]
[in a new window]

  		FIG. 2. F-actin dissociation in OnM{phi}s. (A) Time-lapse video microscopy of RAW 264.7 cells forming an OnM{phi} 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. OnM{phi}s are indicated by arrowheads. Samples were stained for F-actin (red) (B to D), Salmonella (green) (B to D), and CD18 (blue) (B), ß-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 {chi}3306 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.

 
Using confocal microscopy, we observed that while F-actin was located beneath the plasma membrane in uninfected macrophages, OnM{phi}s were uniformly negative for F-actin (Fig. 2B) but remained positive for the macrophage marker CD18 (Fig. 2B). To determine whether other cytoskeletal filaments were reorganized in OnM{phi}s, we stained cells for microtubules and intermediate filament proteins and detected no obvious changes in ß-tubulin and vimentin in OnM{phi}s containing gigantic SCVs (Fig. 2C and D). Taken together, these data indicate that while OnM{phi} formation has little effect on the overall cytoskeletal organization, it is accompanied by F-actin dissociation. Confocal microscopy analysis showed that most F-actin-negative OnM{phi}s contained intracellular Salmonella bacilli (Fig. 2E) but a few OnM{phi}s contained no detectable intracellular bacilli (Fig. 2E), suggesting that intracellular rather than extracellular Salmonella bacilli trigger OnM{phi} formation (for TUNEL staining, see below).

Lack of DNA damage in Salmonella-induced oncosis. By 4 h after infection, more than 20 bacilli were often seen in a single OnM{phi}, as revealed by Giemsa staining (Fig. 3A). Staining also showed that such macrophages lacked chromatin condensation and DNA damage even in the presence of numerous Salmonella bacilli (Fig. 3A).


Figure 3
View larger version (55K):
[in this window]
[in a new window]

  		FIG. 3. Biochemical features of Salmonella-induced OnM{phi}s. (A) Giemsa staining of OnM{phi}s 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), OnM{phi}s 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 OnM{phi}s. Uninfected, Salmonella-infected, and H2O2-treated apoptotic macrophages (control) were examined. OnM{phi}s 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 OnM{phi} (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 OnM{phi}s (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.

 
To reveal biochemical features of Salmonella-induced OnM{phi}s, we first measured the [Ca2+]i using fluorescent imaging. Both OnM{phi}s and apoptotic cells showed higher [Ca2+]i than uninfected cells, but there was no significant difference between oncotic and H2O2-treated apoptotic macrophages (Fig. 3B). Since Salmonella cells produce proapoptotic effectors, such as SipB (5, 8, 29, 44), we measured caspase-1 and caspase-3 activities and found that OnM{phi}s with motile Salmonella bacilli showed both caspase-1 activity (six of six OnM{phi}s) and caspase-3 activity (eight of nine OnM{phi}s) (Fig. 3C). To determine whether DNA damage occurs in OnM{phi}s, we employed a TUNEL assay. Although we observed TUNEL-positive cells that did not show cellular swelling, OnM{phi}s containing numerous bacilli were TUNEL negative (Fig. 3D), supporting the idea that OnM{phi}s do not undergo apoptosis. Confocal microscopy also revealed both TUNEL-negative OnM{phi}s (Fig. 2E) and TUNEL-positive apoptotic macrophages (Fig. 2E).

To analyze the integrity of the host cell plasma membrane, we stained cells with two fluorescent compounds, calcein AM, which passes through intact membranes and detects esterase activity in the cytosol (green), and ethidium homodimer-1, which stains dead cells with permeable membranes (red). Six of 10 swollen macrophages containing motile Salmonella bacilli showed weak green staining and were either negative for or only faintly stained with ethidium homodimer-1, indicating that their plasma membranes were largely intact (Fig. 3E); the other four cells in this group were stained only with the red stain, indicating that these cells underwent cell death (Fig. 3E). These data indicate that over one-half of the swollen macrophages containing motile bacteria had impermeable membranes and showed esterase activity.

Salmonella motility in OnM{phi}s. Although we observed vigorously moving Salmonella bacilli in OnM{phi}s (Fig. 1A, B, and C; see Movie S1 in the supplemental material), flagellum expression is believed to be suppressed inside host cells (12). To determine whether Salmonella motility is flagellum dependent, we performed immunofluorescence staining with an antiflagellum antibody combined with F-actin and TUNEL staining. Confocal laser microscopy revealed that F-actin- and TUNEL-negative OnM{phi}s contained Salmonella bacilli that stained positive for flagella (Fig. 4A).


Figure 4
View larger version (54K):
[in this window]
[in a new window]

  		FIG. 4. Motility of Salmonella bacilli in OnM{phi}s is driven by flagella. (A) Confocal microscopy of OnM{phi}s (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) OnM{phi} formation by flagellum-deficient Salmonella. RAW 264.7 cells were infected with the {chi}3181-derived mutant lacking flagella ({Delta}fliA mutant). The bars indicate the percentages of OnM{phi}s; 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 {Delta}fliA mutant by time-lapse microscopy in a liquid medium (left panels) and in OnM{phi}s 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 {Delta}fliA Salmonella strains in liquid medium and in OnM{phi}s. 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.

 
To further determine whether Salmonella motility in OnM{phi}s was flagellum driven, we infected RAW 264.7 cells with a Salmonella {Delta}fliA mutant which lacked flagella due to a mutation in an alternative sigma factor required for flagellum synthesis (35). Under the various conditions tested, the efficiency of OnM{phi} formation by the Salmonella {Delta}fliA mutant (Fig. 4B) was not significantly different from that of wild-type Salmonella (Fig. 1E). Nonetheless, we did not observe vigorously moving intracellular Salmonella {Delta}fliA mutant bacilli by light microscopy (Fig. 4C). Consistently, time-lapse video microscopy showed that the maximum velocities of wild-type Salmonella bacilli in culture medium (68 µm/s) and in OnM{phi}s (37 µm/s) were significantly greater than that observed for the Salmonella {Delta}fliA mutant (12 µm/s in culture medium and 3 µm/s in OnM{phi}s) (Fig. 4C and D). We also observed that the intracellular wild-type Salmonella but not the Salmonella {Delta}fliA mutant rebounded from the host cell membrane in OnM{phi}s. These data indicate that a subset of intracellular Salmonella bacilli is flagellated and that their motility is flagellum driven. Furthermore, induction of OnM{phi} formation per se does not require a flagellum.

Exit of Salmonella from OnM{phi}s. We next performed time-lapse video microscopy of RAW 264.7 cells 4 to 6 h postinfection after we separated the cells from extracellular Salmonella bacilli using CD11b-conjugated magnet beads and cultured them in the absence of gentamicin. We observed swollen macrophages, which released wild-type Salmonella from one or a few loci in the plasma membrane (Fig. 5A). We found that one Salmonella bacillus exited from a cell per minute over more than 1 h of observation (Fig. 5B). These data suggest that after F-actin dissociation, some Salmonella bacilli moving at random penetrate the host cell membrane, potentially making the locus more vulnerable to penetration by other bacilli. We determined the number of flagellated Salmonella bacilli in each host cell 6 h after infection using antisera against flagella. In contrast to the numerous Salmonella bacilli detected by Giemsa staining 4 h after infection (Fig. 3A), most (68%) swollen macrophages lacking F-actin contained no or fewer than six flagellated Salmonella bacilli 6 h after infection (Fig. 5C). This discrepancy may have been due to degradation of intracellular Salmonella in OnM{phi}s, to the presence of flagellum-free Salmonella not detectable by this assay, or to the escape of flagellated Salmonella bacilli from host cells.


Figure 5
View larger version (26K):
[in this window]
[in a new window]

  		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 ({Delta}fliA). 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.

 
To determine whether intracellular Salmonella bacilli escape from host macrophages in a flagellum-dependent manner, we observed RAW 264.7 cells infected with the wild-type strain or the Salmonella {Delta}fliA mutant lacking flagella for an extended period of time in the absence of gentamicin (46). Eight hours after infection with wild-type Salmonella, most macrophages lacked motile intracellular bacteria. By contrast, the Salmonella {Delta}fliA mutant remained abundant in host cells (Fig. 5D). This observation strongly suggests that after inducing OnM{phi} formation, intracellular Salmonella bacilli exit from the host cell in a flagellum-dependent manner.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we showed that intracellular Salmonella bacilli escape from macrophages after induction of OnM{phi} formation (Fig. 6). Salmonella-induced OnM{phi}s have three attributes: (i) F-actin is dissociated beneath the cell membrane, (ii) they are TUNEL negative, and (iii) they often contain abundant, highly motile, flagellated Salmonella bacilli. OnM{phi}s in which there is vigorous movement of Salmonella bacilli in gigantic SCVs are still viable because they preserve plasma membrane impermeability. Later, flagellated Salmonella bacilli escape intermittently from the host cell over an extended period of time.


Figure 6
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 6. Model for escape of Salmonella from OnM{phi}s. Salmonella bacilli are confined to SCVs after they are engulfed by macrophages. Some infected cells undergo apoptosis, pyroptosis, or autophagy. Other infected cells become OnM{phi}s (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.

 
Oncosis is defined as eukaryotic cell death accompanied by cellular swelling (15, 39). Bacteria such as Shigella flexneri, Pseudomonas aeruginosa, and Brucella abortus reportedly induce host cell oncosis (10, 14, 45, 47). Salmonella-induced delayed cell death is also associated with enlarged macrophages (5, 8, 22).

Although the mechanism of oncosis is unclear, it has been suggested that depleted ATP or increased levels of intracellular calcium ions lead to inefficient functioning of ion channels and resultant cellular swelling (56). Salmonella-induced oncosis is characterized by F-actin dissociation and is flagellum independent. It is currently not known whether Salmonella induces oncosis through ATP depletion or by acting directly on F-actin. Salmonella expresses various proteins functioning in F-actin regulation during host cell invasion (17, 26, 27, 37, 43, 52, 60). In addition, deletion of SpvB (Salmonella plasmid virulence B), which is secreted by intracellular Salmonella bacilli, exhibits ADP-ribosyltransferase activity, and modifies actin (34, 37, 54), resulted in reduced OnM{phi} formation in vitro and impaired intracellular proliferation of Salmonella and survival in mice (unpublished observations). Additional studies are needed to determine precise roles of SpvB and other F-actin regulatory proteins in induction of macrophage oncosis. Activation of macrophages by IFN-{gamma} enhanced Salmonella-induced oncosis but not the proportion of OnM{phi}s containing motile bacteria, presumably due to the increased bactericidal activity in host cells. Therefore, OnM{phi}s packed with motile Salmonella bacilli might be formed only during a narrow window of survival in activated host macrophages.

OnM{phi}s exhibited caspase-1 and -3 activities. However, Salmonella-induced OnM{phi}s were not TUNEL positive, suggesting that Salmonella-induced OnM{phi}s do not undergo DNA damage characteristic of apoptosis. Similarly, in Pseudomonas-induced oncosis, host cells also do not exhibit DNA damage (10).

Our data indicate that intracellular motility of Salmonella is flagellum driven. The velocity of intracellular wild-type Salmonella (37 µm/s) was much greater than that of Listeria, Shigella, or Rickettsia due to formation of "comet tails," which require actin polymerization (up to 0.5 µm/s) (9, 19, 49, 55) or microtubule motors (0.02 µm/s) (21). Salmonella {Delta}fliA mutants lacking flagella also showed significantly reduced motility outside and inside host cells. Third, intracellular Salmonella bacteria in OnM{phi}s were positive for an antiflagellum antibody, although we cannot exclude the possibility that flagella were detached from Salmonella cells (38). Finally, highly motile Salmonella bacilli escaped from host cells, while flagellumless {Delta}fliA mutants did not.

Intracellular flagellation was unanticipated, since it has been reported that intracellular Salmonella bacilli down-regulate flagellum synthesis (12). Salmonella bacilli in OnM{phi}s do not retain flagella through the early stages of infection; we observed that most intracellular Salmonella bacilli were flagellum negative 2 h after infection (data not shown). However, some bacilli were flagellum positive 4 h later, suggesting that flagella are regenerated. We cannot, however, exclude the possibility that some Salmonella bacilli retain flagella throughout infection. Interestingly, flagellum reexpression by intracellular bacteria has also been reported in Legionella (6). Since flagellated Salmonella bacilli exit from host cells by 8 h after infection, it is likely that Salmonella bacilli inside OnM{phi}s down-regulate genes required for flagellum synthesis, as has been reported previously (12), but then up-regulate them prior to exiting from host cells.

Our results indicate that Salmonella induces macrophage oncosis and accumulates in OnM{phi}s, which is followed by intermittent escape of Salmonella bacilli from host cells via flagella. As escape of phagocytosed bacteria from macrophages is critical for establishment of infectious diseases caused by intracellular pathogens, investigation of the possible link between pathogenesis and mechanisms by which pathogens escape from macrophages is warranted.


   ACKNOWLEDGMENTS
 
We thank Kazuhiro Kutsukake for providing Salmonella LT2 strain KK2091, Kohei Honma for technical help with analyzing bacterial velocity, and Hiroshi Handa for support. We also thank Chihiro Sasakawa, Fidel Zavala, Tetsuya Iida, Neelanjan Ray, and Elise Lamar for critical reading of the manuscript.

This work was supported by Grant-in-Aid for Scientific Research (C) 17590400 and "High-Tech Research Center" Project 020610044 to G.S., by a Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects, and by the 21st Century COE Program at Keio University from the MEXT of the Japanese Government.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone: (81) 3 3353 1211. Fax: (81) 3 5360 1508. E-mail: matsuo{at}sc.itc.keio.ac.jp Back

{triangledown} Published ahead of print on 14 September 2007. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Adams, P., R. Fowler, N. Kinsella, G. Howell, M. Farris, P. Coote, and C. D. O'Connor. 2001. Proteomic detection of PhoPQ- and acid-mediated repression of Salmonella motility. Proteomics 1:597-607.[CrossRef][Medline]
  2. Bharti, A. C., Y. Takada, and B. B. Aggarwal. 2005. PARP cleavage and caspase activity to assess chemosensitivity. Methods Mol. Med. 111:69-78.[Medline]
  3. Boucrot, E., T. Henry, J. P. Borg, J. P. Gorvel, and S. Méresse. 2005. The intracellular fate of Salmonella depends on the recruitment of kinesin. Science 308:1174-1178.[Abstract/Free Full Text]
  4. Brennan, M. A., and B. T. Cookson. 2000. Salmonella induces macrophage death by caspase-1-dependent necrosis. Mol. Microbiol. 38:31-40.[CrossRef][Medline]
  5. Browne, S. H., M. L. Lesnick, and D. G. Guiney. 2002. Genetic requirements for Salmonella-induced cytopathology in human monocyte-derived macrophages. Infect. Immun. 70:7126-7135.[Abstract/Free Full Text]
  6. Byrne, B., and M. S. Swanson. 1998. Expression of Legionella pneumophila virulence traits in response to growth conditions. Infect. Immun. 66:3029-3034.[Abstract/Free Full Text]
  7. Chakravortty, D., I. Hansen-Wester, and M. Hensel. 2002. Salmonella pathogenicity island 2 mediates protection of intracellular Salmonella from reactive nitrogen intermediates. J. Exp. Med. 195:1155-1166.[Abstract/Free Full Text]
  8. Chen, L. M., K. Kaniga, and J. E. Galán. 1996. Salmonella spp. are cytotoxic for cultured macrophages. Mol. Microbiol. 21:1101-1115.[CrossRef][Medline]
  9. Dabiri, G. A., J. M. Sanger, D. A. Portnoy, and F. S. Southwick. 1990. Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly. Proc. Natl. Acad. Sci. USA 87:6068-6072.[Abstract/Free Full Text]
  10. Dacheux, D., B. Toussaint, M. Richard, G. Brochier, J. Croize, and I. Attree. 2000. Pseudomonas aeruginosa cystic fibrosis isolates induce rapid, type III secretion-dependent, but ExoU-independent, oncosis of macrophages and polymorphonuclear neutrophils. Infect. Immun. 68:2916-2924.[Abstract/Free Full Text]
  11. Detweiler, C. S., D. M. Monack, I. E. Brodsky, H. Mathew, and S. Falkow. 2003. virK, somA and rcsC are important for systemic Salmonella enterica serovar Typhimurium infection and cationic peptide resistance. Mol. Microbiol. 48:385-400.[CrossRef][Medline]
  12. Eriksson, S., S. Lucchini, A. Thompson, M. Rhen, and J. C. Hinton. 2003. Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica. Mol. Microbiol. 47:103-118.[CrossRef][Medline]
  13. Ernst, R. K., T. Guina, and S. I. Miller. 2001. Salmonella typhimurium outer membrane remodeling: role in resistance to host innate immunity. Microbes Infect. 3:1327-1334.[CrossRef][Medline]
  14. Fernandez-Prada, C. M., D. L. Hoover, B. D. Tall, A. B. Hartman, J. Kopelowitz, and M. M. Venkatesan. 2000. Shigella flexneri IpaH(7.8) facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages. Infect. Immun. 68:3608-3619.[Abstract/Free Full Text]
  15. Fink, S. L., and B. T. Cookson. 2005. Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect. Immun. 73:1907-1916.[Free Full Text]
  16. Fratti, R. A., J. Chua, I. Vergne, and V. Deretic. 2003. Mycobacterium tuberculosis glycosylated phosphatidylinositol causes phagosome maturation arrest. Proc. Natl. Acad. Sci. USA 100:5437-5442.[Abstract/Free Full Text]
  17. Fu, Y., and J. E. Galán. 1999. A Salmonella protein antagonizes Rac-1 and Cdc42 to mediate host-cell recovery after bacterial invasion. Nature 401:293-297.[CrossRef][Medline]
  18. Garcia-del Portillo, F., M. B. Zwick, K. Y. Leung, and B. B. Finlay. 1993. Salmonella induces the formation of filamentous structures containing lysosomal membrane glycoproteins in epithelial cells. Proc. Natl. Acad. Sci. USA 90:10544-10548.[Abstract/Free Full Text]
  19. Gouin, E., H. Gantelet, C. Egile, I. Lasa, H. Ohayon, V. Villiers, P. Gounon, P. J. Sansonetti, and P. Cossart. 1999. A comparative study of the actin-based motilities of the pathogenic bacteria Listeria monocytogenes, Shigella flexneri and Rickettsia conorii. J. Cell Sci. 112:1697-1708.[Abstract]
  20. Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440-3450.[Abstract/Free Full Text]
  21. Guignot, J., E. Caron, C. Beuzon, C. Bucci, J. Kagan, C. Roy, and D. W. Holden. 2004. Microtubule motors control membrane dynamics of Salmonella-containing vacuoles. J. Cell Sci. 117:1033-1045.[Abstract/Free Full Text]
  22. Guilloteau, L. A., T. S. Wallis, A. V. Gautier, S. MacIntyre, D. J. Platt, and A. J. Lax. 1996. The Salmonella virulence plasmid enhances Salmonella-induced lysis of macrophages and influences inflammatory responses. Infect. Immun. 64:3385-3393.[Abstract]
  23. Guiney, D. G. 2005. The role of host cell death in Salmonella infections. Curr. Top. Microbiol. Immunol. 289:131-150.[Medline]
  24. Gulig, P. A., A. L. Caldwell, and V. A. Chiodo. 1992. Identification, genetic analysis and DNA sequence of a 7.8-kb virulence region of the Salmonella typhimurium virulence plasmid. Mol. Microbiol. 6:1395-1411.[Medline]
  25. Gulig, P. A., and R. Curtiss III. 1987. Plasmid-associated virulence of Salmonella typhimurium. Infect. Immun. 55:2891-2901.[Abstract/Free Full Text]
  26. Hardt, W. D., L. M. Chen, K. E. Schuebel, X. R. Bustelo, and J. E. Galán. 1998. S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells. Cell 93:815-826.[CrossRef][Medline]
  27. Hayward, R. D., and V. Koronakis. 1999. Direct nucleation and bundling of actin by the SipC protein of invasive Salmonella. EMBO J. 18:4926-4934.[CrossRef][Medline]
  28. Hernandez, L. D., M. Pypaert, R. A. Flavell, and J. E. Galán. 2003. A Salmonella protein causes macrophage cell death by inducing autophagy. J. Cell Biol. 163:1123-1131.[Abstract/Free Full Text]
  29. Hersh, D., D. M. Monack, M. R. Smith, N. Ghori, S. Falkow, and A. Zychlinsky. 1999. The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1. Proc. Natl. Acad. Sci. USA 96:2396-2401.[Abstract/Free Full Text]
  30. 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.[Abstract/Free Full Text]
  31. Hueffer, K., and J. E. Galán. 2004. Salmonella-induced macrophage death: multiple mechanisms, different outcomes. Cell. Microbiol. 6:1019-1025.[CrossRef][Medline]
  32. Jesenberger, V., K. J. Procyk, J. Yuan, S. Reipert, and M. Baccarini. 2000. Salmonella-induced caspase-2 activation in macrophages: a novel mechanism in pathogen-mediated apoptosis. J. Exp. Med. 192:1035-1046.[Abstract/Free Full Text]
  33. Kubota, T., Y. Shindo, K. Tokuno, H. Komatsu, H. Ogawa, S. Kudo, Y. Kitamura, K. Suzuki, and K. Oka. 2005. Mitochondria are intracellular magnesium stores: investigation by simultaneous fluorescent imagings in PC12 cells. Biochim. Biophys. Acta 1744:19-28.[Medline]
  34. Kurita, A., H. Gotoh, M. Eguchi, N. Okada, S. Matsuura, H. Matsui, H. Danbara, and Y. Kikuchi. 2003. Intracellular expression of the Salmonella plasmid virulence protein, SpvB, causes apoptotic cell death in eukaryotic cells. Microb. Pathog. 35:43-48.[CrossRef][Medline]
  35. Kutsukake, K., Y. Ohya, S. Yamaguchi, and T. Iino. 1988. Operon structure of flagellar genes in Salmonella typhimurium. Mol. Gen. Genet. 214:11-15.[CrossRef][Medline]
  36. Lee, A. K., C. S. Detweiler, and S. Falkow. 2000. OmpR regulates the two-component system SsrA-SsrB in Salmonella pathogenicity island 2. J. Bacteriol. 182:771-781.[Abstract/Free Full Text]
  37. Lesnick, M. L., N. E. Reiner, J. Fierer, and D. G. Guiney. 2001. The Salmonella spvB virulence gene encodes an enzyme that ADP-ribosylates actin and destabilizes the cytoskeleton of eukaryotic cells. Mol. Microbiol. 39:1464-1470.[CrossRef][Medline]
  38. Lyons, S., L. Wang, J. E. Casanova, S. V. Sitaraman, D. Merlin, and A. T. Gewirtz. 2004. Salmonella typhimurium transcytoses flagellin via an SPI2-mediated vesicular transport pathway. J. Cell Sci. 117:5771-5780.[Abstract/Free Full Text]
  39. Majno, G., and I. Joris. 1995. Apoptosis, oncosis, and necrosis. An overview of cell death. Am. J. Pathol 146:3-15.[Abstract]
  40. Maruyama, K., G. Sano, N. Ray, Y. Takada, and K. Matsuo. 2007. c-Fos-deficient mice are susceptible to Salmonella enterica serovar Typhimurium infection. Infect. Immun. 75:1520-1523.[Abstract/Free Full Text]
  41. Matsui, H., T. Kawakami, S. Ishikawa, H. Danbara, and P. A. Gulig. 2000. Constitutively expressed phoP inhibits mouse-virulence of Salmonella typhimurium in an Spv-dependent manner. Microbiol. Immunol. 44:447-454.[Medline]
  42. Matsui, H., M. Suzuki, Y. Isshiki, C. Kodama, M. Eguchi, Y. Kikuchi, K. Motokawa, A. Takaya, T. Tomoyasu, and T. Yamamoto. 2003. Oral immunization with ATP-dependent protease-deficient mutants protects mice against subsequent oral challenge with virulent Salmonella enterica serovar Typhimurium. Infect. Immun. 71:30-39.[Abstract/Free Full Text]
  43. McGhie, E. J., R. D. Hayward, and V. Koronakis. 2001. Cooperation between actin-binding proteins of invasive Salmonella: SipA potentiates SipC nucleation and bundling of actin. EMBO J. 20:2131-2139.[CrossRef][Medline]
  44. Monack, D. M., B. Raupach, A. E. Hromockyj, and S. Falkow. 1996. Salmonella typhimurium invasion induces apoptosis in infected macrophages. Proc. Natl. Acad. Sci. USA 93:9833-9838.[Abstract/Free Full Text]
  45. Nonaka, T., A. Kuwae, C. Sasakawa, and S. Imajoh-Ohmi. 1999. Shigella flexneri YSH6000 induces two types of cell death, apoptosis and oncosis, in the differentiated human monoblastic cell line U937. FEMS Microbiol. Lett. 174:89-95.[CrossRef][Medline]
  46. Ohya, S., H. Xiong, Y. Tanabe, M. Arakawa, and M. Mitsuyama. 1998. Killing mechanism of Listeria monocytogenes in activated macrophages as determined by an improved assay system. J. Med. Microbiol. 47:211-215.[Abstract]
  47. Pei, J., J. E. Turse, Q. Wu, and T. A. Ficht. 2006. Brucella abortus rough mutants induce macrophage oncosis that requires bacterial protein synthesis and direct interaction with the macrophage. Infect. Immun. 74:2667-2675.[Abstract/Free Full Text]
  48. Ray, N., M. Kuwahara, Y. Takada, K. Maruyama, T. Kawaguchi, H. Tsubone, H. Ishikawa, and K. Matsuo. 2006. c-Fos suppresses systemic inflammatory response to endotoxin. Int. Immunol. 18:671-677.[Abstract/Free Full Text]
  49. Robbins, J. R., A. I. Barth, H. Marquis, E. L. de Hostos, W. J. Nelson, and J. A. Theriot. 1999. Listeria monocytogenes exploits normal host cell processes to spread from cell to cell. J. Cell Biol. 146:1333-1350.[Abstract/Free Full Text]
  50. Schmieger, H. 1972. Phage P22-mutants with increased or decreased transduction abilities. Mol. Gen. Genet. 119:75-88.[CrossRef][Medline]
  51. Shea, J. E., M. Hensel, C. Gleeson, and D. W. Holden. 1996. Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 93:2593-2597.[Abstract/Free Full Text]
  52. Stender, S., A. Friebel, S. Linder, M. Rohde, S. Mirold, and W. D. Hardt. 2000. Identification of SopE2 from Salmonella typhimurium, a conserved guanine nucleotide exchange factor for Cdc42 of the host cell. Mol. Microbiol. 36:1206-1221.[CrossRef][Medline]
  53. Swanson, M. S., and R. R. Isberg. 1995. Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect. Immun. 63:3609-3620.[Abstract]
  54. Tezcan-Merdol, D., T. Nyman, U. Lindberg, F. Haag, F. Koch-Nolte, and M. Rhen. 2001. Actin is ADP-ribosylated by the Salmonella enterica virulence-associated protein SpvB. Mol. Microbiol. 39:606-619.[CrossRef][Medline]
  55. Theriot, J. A., T. J. Mitchison, L. G. Tilney, and D. A. Portnoy. 1992. The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. Nature 357:257-260.[CrossRef][Medline]
  56. Trump, B. F., I. K. Berezesky, S. H. Chang, and P. C. Phelps. 1997. The pathways of cell death: oncosis, apoptosis, and necrosis. Toxicol. Pathol. 25:82-88.[Medline]
  57. Uchiya, K., M. A. Barbieri, K. Funato, A. H. Shah, P. D. Stahl, and E. A. Groisman. 1999. A Salmonella virulence protein that inhibits cellular trafficking. EMBO J. 18:3924-3933.[CrossRef][Medline]
  58. Vazquez-Torres, A., Y. Xu, J. Jones-Carson, D. W. Holden, S. M. Lucia, M. C. Dinauer, P. Mastroeni, and F. C. Fang. 2000. Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase. Science 287:1655-1658.[Abstract/Free Full Text]
  59. Yoshida, S., Y. Handa, T. Suzuki, M. Ogawa, M. Suzuki, A. Tamai, A. Abe, E. Katayama, and C. Sasakawa. 2006. Microtubule-severing activity of Shigella is pivotal for intercellular spreading. Science 314:985-989.[Abstract/Free Full Text]
  60. Zhou, D., L. M. Chen, L. Hernandez, S. B. Shears, and J. E. Galán. 2001. A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host cell actin cytoskeleton rearrangements and bacterial internalization. Mol. Microbiol. 39:248-259.[CrossRef][Medline]

Journal of Bacteriology, November 2007, p. 8224-8232, Vol. 189, No. 22
0021-9193/07/$08.00+0     doi:10.1128/JB.00898-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Other Versions of this Article:
JB.00898-07v1
189/22/8224    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sano, G.-i.
Right arrow Articles by Matsuo, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sano, G.-i.
Right arrow Articles by Matsuo, K.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Appl. Environ. Microbiol. Infect. Immun. Eukaryot. Cell
Mol. Cell. Biol. J. Virol. Microbiol. Mol. Biol. Rev.
ALL ASM JOURNALS