• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Oct 12, 2010; 107(41): 17733–17738.
Published online Sep 27, 2010. doi:  10.1073/pnas.1006098107
PMCID: PMC2955089
Microbiology

Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia

Abstract

Salmonella enterica is an intracellular bacterial pathogen that resides and proliferates within a membrane-bound vacuole in epithelial cells of the gut and gallbladder. Although essential to disease, how Salmonella escapes from its intracellular niche and spreads to secondary cells within the same host, or to a new host, is not known. Here, we demonstrate that a subpopulation of Salmonella hyperreplicating in the cytosol of epithelial cells serves as a reservoir for dissemination. These bacteria are transcriptionally distinct from intravacuolar Salmonella. They are induced for the invasion-associated type III secretion system and possess flagella; hence, they are primed for invasion. Epithelial cells laden with these cytosolic bacteria are extruded out of the monolayer, releasing invasion-primed and -competent Salmonella into the lumen. This extrusion mechanism is morphologically similar to the process of cell shedding required for turnover of the intestinal epithelium. In contrast to the homeostatic mechanism, however, bacterial-induced extrusion is accompanied by an inflammatory cell death characterized by caspase-1 activation and the apical release of IL-18, an important cytokine regulator of gut inflammation. Although epithelial extrusion is obviously beneficial to Salmonella for completion of its life cycle, it also provides a mechanistic explanation for the mucosal inflammation that is triggered during Salmonella infection of the gastrointestinal and biliary tracts.

Keywords: caspase-1, epithelial cells, flagella, IL-18, type III secretion

Intracellular pathogens reside either within a membrane-bound vacuole or freely within the host cell cytosol. However, regardless of their specific lifestyle, three distinct steps are common to their infectious cycle: the ability to enter host cells, their establishment of an intracellular niche, and their escape from the host cell (1). Entry and intracellular survival are critical virulence stages for these pathogens, but exit from the infected cell is essential for dissemination and transmission to other hosts. Although considerable progress has been made in elucidating the first two facets of this cycle, the mechanism by which intracellular pathogens escape from host cells has been comparably neglected.

The Gram-negative bacterium Salmonella enterica causes a wide range of food- and water-borne diseases ranging from self-limiting gastroenteritis to systemic typhoid fever in both humans and animals. In enteric infections, Salmonella preferentially targets the single layer of polarized columnar epithelial cells lining the surface of the gastrointestinal tract (24), triggering an extensive inflammatory response. After invading epithelial cells from the apical side, Salmonella resides and replicates within a membrane-bound vacuole, known as the Salmonella-containing vacuole (SCV). Symptomatic and asymptomatic infections are characterized by the fecal shedding of bacteria (5, 6), suggesting that Salmonella escapes from its intracellular niche back into the gut lumen as part of its infectious cycle. Here, we report that Salmonella exits from polarized epithelia by coopting a mechanism normally used by the host to remove senescent cells from the mucosal epithelium.

Results and Discussion

WT Salmonella Hyperreplicates in the Cytosol of Epithelial Cells.

Human colonic epithelial cells (C2BBe1, a subclone of Caco-2) grown on filters were used as a model polarized monolayer to examine the infectious cycle of Salmonella. Confocal microscopy analysis of infected monolayers revealed two distinct populations of proliferating bacteria following the onset of replication ≥4 h postinfection (p.i.) (Fig. 1A and Fig. S1A). Compared with an average doubling time of ≥95 min for the total population (Fig. S1A), some bacteria were replicating at a much faster rate, with a doubling time of ~20 min (Movie S1). There was a temporal increase in the incidence of these “hyperreplicating” Salmonella (defined as >50 bacteria per cell) (Fig. 1 A and B). By 10 h p.i., 11 ± 4.2% of infected cells contained hyperreplicating bacteria (Fig. 1A). A similar phenotype was previously described for a Salmonella sifA mutant, which hyperreplicates in the host cell cytosol because of a defect in maintaining vacuolar integrity (7). We therefore assessed whether the hyperreplicating WT Salmonella we observed in polarized epithelial cells are also free in the cytosol. Confocal microscopy indicated that many of these bacteria were not in a lysosome-associated membrane protein 1 (LAMP1)-positive compartment (Fig. 1B and Fig. S1B), and thus not in a mature SCV (8). Selective membrane permeabilization followed by immunostaining with polyclonal anti-Salmonella LPS antibody revealed that at least one-third of the hyperreplicating bacteria are cytosolic (Fig. 1C and Fig. S2). These experiments demonstrate that WT Salmonella can replicate in a vacuole and the cytosol in epithelial cells, but they proliferate more efficiently in the cytosolic environment (7, 9, 10).

Fig. 1.
Infected epithelial cells contain two distinct populations of replicating Salmonella. (A) Polarized C2BBe1 monolayers were infected with WT Salmonella constitutively expressing mCherry. Monolayers were fixed at the indicated times and immunostained for ...

Cytosolic Salmonella Are Invasion-Primed.

We hypothesized that the two distinct intracellular environments, intravacuolar and cytosolic, would differentially influence the expression of bacterial virulence genes. To assess this, we used a plasmid-derived transcriptional fusion assay based on destabilized GFP(LVA). Promoters were selected from well-characterized genes in each of the three type III secretion systems (T3SSs): PfliC-gfp[LVA] (flagellar T3SS), PprgH-gfp[LVA] (T3SS1), and PssaG-gfp[LVA] (T3SS2) (11). The number of fluorescent bacteria was monitored with time. Under the infection conditions used here, efficient invasion requires both T3SS1 and flagellar-based motility (1113) (Fig. S1A). This single-cell assay confirmed that both T3SS1 (PprgH-gfp[LVA]) and flagella (PfliC-gfp[LVA]) were rapidly down-regulated after bacterial internalization (11, 14, 15) (Fig. S1C). Surprisingly, these virulence factors were not completely inactivated (Fig. 2 and Fig. S1C). At 10 h p.i., ~6% of infected cells contained fluorescent PprgH-GFP[LVA] bacteria (3.8 ± 1.2% of the total bacterial population) (Fig. 2 and S1C). Strikingly, these T3SS1-induced bacteria were almost exclusively found in cells containing hyperreplicating bacteria (Fig. 2A) and associated with flagella (Fig. 2D). As expected, T3SS2 was induced intracellularly (16) (Fig. S1C); fluorescent PssaG-GFP[LVA] bacteria were not detected until >2 h p.i., and 32 ± 6.6% of the bacteria were GFP-positive by 10 h p.i. (Figs. S1C and S3). At 8 h p.i., ~60% of the T3SS1-induced bacteria were cytosolic and 23 ± 6% were in LAMP1-positive SCVs (Fig. 2 B, C, and E). By contrast, the T3SS2-induced bacteria were intravacuolar (91 ± 6% LAMP1-positive) and typically found in cells containing 5–20 bacteria (Fig. 2E and Fig. S3). Using live cell imaging, the motility of intracellular Salmonella was assessed at 8 h p.i. (Fig. 2F). Consistent with flagellin (FliC) expression (Fig. 2D), a subset of T3SS1-induced bacteria was motile (Fig. 2F and Movie S2). This population moved at speeds consistent with flagellar-based motility (4–15 μm/s) (17), whereas T3SS2-induced bacteria were immobile (Fig. 2F and Movie S2). Hence, there are at least two transcriptionally distinct intracellular populations of replicating bacteria in epithelial cells: T3SS2-induced intravacuolar bacteria and T3SS1-induced flagellated bacteria that are cytosolic.

Fig. 2.
Flagella and the invasion-associated T3SS1 are expressed by cytosolic bacteria late during infection. (AD) Polarized C2BBe1 monolayers were infected with WT Salmonella carrying a plasmid that expresses destabilized GFP [GFP(LVA)] under the control ...

Invasion-Primed Salmonella Are Released into the Lumen by Extruding Cells.

Coincident with the onset and kinetics of hyperreplication, bacteria-laden cells extruding toward the apical side were observed by EM (Fig. 3 A and B). Bacteria in these extruded cells were not surrounded by a vacuolar membrane [Fig. 3B (ii)], in agreement with the confocal microscopy data for hyperreplicating bacteria (Figs. 1C and and22E). By contrast, SCV membranes were readily apparent around replicating bacteria in cells within the monolayer (Fig. 3C). To examine whether extrusion of infected epithelial cells occurs in vivo, we used a mouse model in which Salmonella rapidly breaches the intestinal barrier and spreads systemically to various tissues, including the mucosal epithelium lining the gallbladder. In this murine infection model, Salmonella causes inflammation similar to cholecystitis seen in humans during acute typhoid fever (18). We observed both free bacteria and bacteria-laden epithelial cells in the gallbladder lumen (Fig. 3 D and E). In agreement with our observations, sloughing of enterocytes laden with Salmonella from villus tips has also been reported in rabbit ileal loop studies (2). Therefore, extrusion of Salmonella-infected epithelial cells is evident in both enteric and systemic infections.

Fig. 3.
Apical extrusion of infected cells from mucosal epithelium in vitro and in vivo. (A) SEM of the apical surface of an infected monolayer. Polarized C2BBe1 cells were infected with WT Salmonella, and at 10 h p.i., samples were fixed and processed for SEM. ...

Salmonella-associated extrusion resembles cell extrusion involved in the rapid turnover of polarized epithelial cells in the gut. This process occurs when neighboring cells contract to push a dying cell out of the monolayer and is characterized by reorganization of adherens and tight junctions to maintain the integrity of the epithelial monolayer (1922). Here, we observed similar actin contractile rings and tight junction “rosettes” at the base of extruding Salmonella-infected cells (Fig. S4 and Movie S3), suggesting that the Salmonella-associated and homeostatic extrusion events occur via similar cell biological processes.

The incidence of extrusion was significantly increased on infection: 10 ± 2.7% of cells containing Salmonella showed evidence of extrusion at 10 h p.i. compared with only 0.85 ± 0.89% of uninfected cells (Fig. 4A). Furthermore, cells containing T3SS1-induced Salmonella were more likely to be extruded than those containing T3SS2-induced bacteria (20 ± 4.7% vs. 2.4 ± 0.9%; Fig. 4 A and B). In infected gallbladders, T3SS1-induced bacteria were found within epithelial cells lining the gallbladder at 4 d p.i. and did not immunostain for FliC (Fig. S5). By 5 d p.i., flagellated T3SS1-induced bacteria were predominantly found within cells that had been sloughed into the lumen or were free in the lumen (Fig. 4C and Fig. S5). Altogether, these data imply a strong correlation between T3SS1 induction and Salmonella-induced extrusion in vitro and in vivo. To address the invasion competence of these bacteria, we developed a secondary infection assay (Fig. S6A). Consistent with the onset of cell extrusion from the polarized monolayer, significant numbers of bacteria could be recovered from a naive population of epithelial cells from 6 h p.i. and increasing thereafter. This demonstrates that Salmonella released from extruded cells are invasion-primed and -competent.

Fig. 4.
Extruding epithelial cells contain invasion-primed Salmonella. (A) Quantification of extrusion. Polarized C2BBe1 monolayers were infected with Salmonella constitutively expressing mCherry or carrying destabilized GFP reporters for T3SS1 (PprgH-GFP[LVA]) ...

Extruding Cells Undergo Inflammatory Cell Death.

During gut homeostasis, epithelial cells are shed into the lumen as a result of anoikis, a form of apoptosis characterized by activated caspase-2, -3, and -9 but not caspase-1 or -8 (2326). Because the Salmonella-laden extruding cells also showed structural features typical of cell death both in vivo and in vitro (Fig. 3 B, D, and E), we next assayed for caspase activity. Epithelial monolayers were incubated with cell-permeable fluorescent probes that bind irreversibly to activated caspases. Uninfected extruding cells were positive for active caspase-3/-7 (74 ± 8.1%) but not for active caspase-1 (21 ± 9.4%), as previously described (27) (Fig. 5B). By contrast, the majority of infected extruding cells were positive for both active caspase-1 (83 ± 4.5%) and caspase-3/-7 (85 ± 7.3%) (Fig. 5 A and B). Homeostatic extrusion of epithelial cells is not prevented by general caspase inhibition (21). Addition of a caspase-1 inhibitor did not block, but significantly decreased, extrusion of infected cells (Fig. 4A), suggesting that the signal for homeostatic and bacterial-induced extrusion precedes caspase activation.

Fig. 5.
Salmonella-infected extruding epithelial cells undergo inflammatory cell death. (A) Confocal image of an infected extruding cell positive for active caspase-1. Polarized C2BBe1 monolayers were infected with WT Salmonella constitutively expressing mCherry ...

Caspase-1–dependent programmed cell death, also known as pyroptosis, is characterized by pore formation in the plasma membrane, followed by cell swelling and lysis, and the proteolytic processing of proinflammatory cytokines, leading to the secretion of mature active IL-1β and IL-18 (28). To monitor plasma membrane integrity, infected cells were incubated with SYTOX Orange nucleic acid dye, which can only enter cells with a compromised plasma membrane, and Hoechst 33342, a cell-permeable nuclear stain. Salmonella-infected extruding cells were positive for both dyes, indicating plasma membrane rupture, whereas neighboring cells within the monolayer stained only with Hoechst 33342 (Fig. 5C). We next quantified the apical and basolateral release of cytokines from infected C2BBe1 cells. Although IL-1β release was below the limits of detection (0.8 pg/mL), we observed a steady temporal increase in the release of IL-18, which was restricted to the apical side of monolayers (Fig. 5D) and dependent on caspase-1 and -3 (Fig. 5E). Collectively, these data highlight a clear difference in the activated cell death program between Salmonella-induced and homeostatic extrusion.

We have demonstrated that extruding Salmonella-infected cells undergo inflammatory cell death. A complex but still largely unexplained pathological feature of salmonelloses is an overwhelming inflammatory response. We propose that bacterial-induced extrusion provides one mechanistic explanation for the pathogenesis of mucosal inflammation during Salmonella infections of the intestine and gallbladder. In support of our data, activation of IL-18 in porcine intestinal mucosa has been reported for S. enterica serovar Choleraesuis infections (29). Interestingly, in inflammatory bowel diseases, chronic inflammation is also associated with elevated IL-18 levels (3032). Therefore, caspase-1–dependent IL-18 production by intestinal epithelial cells might prove to be a mediator of mucosal inflammation associated with both autoimmune disorders (33, 34) and bacterial infections.

Conceptually, an increased turnover of mucosal epithelium provides the host with an ideal defense mechanism against infection. Indeed, it has been implicated as a protective mechanism in the gut against both bacteria and parasites (35, 36). However, we believe that Salmonella takes advantage of this process as a unique means of bacterial egress. Critical to this is the finding that a population of Salmonella is cytosolic and expresses the virulence genes required for invasion. We hypothesize that a vacuole maturation defect leads to the cytosolic release of a small, but significant, fraction of bacteria. The nutrient-rich cytosol supports a high bacterial replication rate and reprograms virulence gene expression toward invasion. The cytosolic load of bacteria is sensed by the host cell, leading to inflammatory cell death and extrusion, releasing the invasion-primed Salmonella into the lumen of the gastrointestinal and biliary tracts. Escape into the lumen allows Salmonella to infect secondary cells rapidly, and may also contribute to host-to-host transmission. Thus, by subverting a host-dependent cell turnover event, Salmonella completes its infectious cycle (Fig. S6B). Given the prevalence of mucosal-dwelling pathogens, other pathogens may also use this host cell process as an exit strategy.

Materials and Methods

Bacterial Strains and Plasmids.

WT S. enterica serovar Typhimurium (S. Typhimurium) SL1344 (37) and ΔSPI2::kan (38), ΔSPI1::kan (15), and flgB::Tn10 mutants (11) have been described previously. For constitutive expression of GFP or mCherry, WT S. Typhimurium was electroporated with pFPV25.1 (39) or pFPV-mCherry (40), respectively. S. Typhimurium carrying the destabilized GFP (GFP[LVA]) (41) reporter plasmids pMPMA3ΔPlac-PprgH-gfp[LVA], pMPMA3ΔPlac-PinvF-gfp[LVA], pMPMA3ΔPlac-PfliC-gfp[LVA], or pMPMA3ΔPlac-PssaG-gfp[LVA] (11) were used to analyze intracellular virulence gene expression.

Infection of Cultured Epithelial Cells with Salmonella.

Cell lines were obtained from the American Type Culture Collection and used at a passage number ≤13 for all experiments. C2BBe1 human colorectal adenocarcinoma cells (CRL-2012), a clone of Caco-2, were maintained in DMEM (Mediatech) containing 10 μg/mL human transferrin (Sigma) and 10% (vol/vol) heat-inactivated FCS (Gibco). HeLa human cervical adenocarcinoma cells were maintained in Eagle's modified Eagle medium (Mediatech) containing 10% (vol/vol) heat-inactivated FCS. Polarized monolayers were established by seeding 2 × 105 C2BBe1 cells in basal seeding medium containing MITO+ serum extender (Becton Dickinson) on BIOCOAT fibrillar collagen 24-well inserts with a 1-μm membrane pore size (Becton Dickinson). After 24 h, the seeding medium was replaced with enterocyte differentiation medium containing MITO+ serum extender. Cells were incubated in differentiation medium for a total of 3 d, replaced with fresh medium each day, until the transepithelial electrical resistance was ≥250 Ω.cm2, as measured using a Millicell Electrical Resistance System (Millipore). The medium was changed to DMEM containing 10% (vol/vol) heat-inactivated FCS (growth medium, GM) before infection.

Preparation of invasive Salmonella and infection of HeLa cells at a multiplicity of infection (MOI) of ~50 were as previously described (42). Polarized monolayers were infected apically at an MOI of ~50–100 for 10 min and then washed three times apically and twice basolaterally in HBSS (Mediatech). Cells were incubated in antibiotic-free GM until 30 min p.i. Thereafter, GM containing 50 μg/mL gentamicin (Sigma) was added for 1 h to kill any remaining extracellular bacteria, followed by GM containing 10 μg/mL gentamicin for the remainder of the experiment to restrict the extracellular growth of bacteria.

Enumeration of Intracellular and Extracellular Bacteria.

For quantification of viable intracellular bacteria, polarized monolayers were washed apically and basolaterally three times with PBS and then lysed in 1 mL of 1% (vol/vol) Triton X-100/0.1% (wt/vol) SDS (TX-100/SDS). Serial dilutions were plated on LB agar plates. Bacterial doubling time was calculated as described previously (43).

To measure the invasion competence of apically released bacteria, we developed a secondary infection protocol. Polarized C2BBe1 monolayers were infected and treated with 50 μg/mL gentamicin to kill extracellular bacteria as described above. At 1.5 h p.i., transwells were inverted onto a monolayer of HeLa cells seeded in six-well plates (2 × 105 cells per well and two transwells per well). Coincubation of C2BBe1 and HeLa cells continued in GM containing 10 μg/mL gentamicin. Polarized monolayers were solubilized in TX-100/SDS as described above. For quantification of the secondary infection, the HeLa cells were washed extensively in HBSS, solubilized in 1 mL of TX-100/SDS, and plated on LB agar plates.

Immunofluorescence Staining.

Infected monolayers were washed twice, apically and basolaterally, with PBS and then fixed apically and basolaterally with 3.5% (wt/vol) paraformaldehyde (PFA) for 20 min at room temperature (RT). Monolayers were then washed apically and basolaterally with PBS, followed by incubation in 75 mM ammonium chloride/20 mM glycine in PBS for 10 min at RT to quench free aldehyde groups. After washing in PBS, monolayers were permeabilized in 10% (vol/vol) normal goat serum/0.1% (wt/vol) saponin in PBS (SS-PBS) for 20 min at RT. Primary and secondary antibodies were diluted in SS-PBS and applied apically and basolaterally in a humid box for 1 h at RT. Filters were excised from transwell supports and placed cell side up on a drop of Prolong Gold antifade reagent (Invitrogen) on a glass slide. Another drop of mounting media was applied to the filter, and a coverslip was placed directly on top. Samples were cured overnight at RT.

Live cell staining was carried out by incubation for 10 min at 37 °C with 2 μg/mL Hoechst 33342 (Invitrogen) to stain nucleic acids or 0.5 μM SYTOX Orange (Invitrogen) to stain nucleic acids in cells with a compromised plasma membrane. For determination of activated caspase-1 and caspase-3/-7, live cells were incubated with FAM-YVAD-FMK or FAM-DEVD-FMK, respectively, in GM for 1 h according to the manufacturer's instruction (Immunochemistry Technologies) before fixation.

To determine whether intracellular bacteria were vacuolar or cytosolic, polarized C2BBe1 cells were infected as described, and at 8 h p.i., the apical plasma membrane was selectively permeabilized using digitonin to allow access of antibody to the cytosol. Briefly, transwells were washed three times in KHM buffer [110 mM potassium acetate, 20 mM Hepes, 2 mM MgCl2 (pH 7.3)] and incubated apically with 150 μg/mL digitonin (Sigma) in KHM buffer for 90 s at RT. Transwells were then immediately washed with KHM buffer. Rabbit polyclonal anti-Salmonella LPS antibody and mouse anti-human GM130 monoclonal antibody were added for 15 min at 37 °C to label cytosolic bacteria and the cytosolic face of the Golgi, respectively. Monolayers were then fixed and quenched as described above, followed by nonselective permeabilization in 0.1% (wt/vol) saponin and 10% (vol/vol) horse serum in PBS for 15 min at RT. Anti-Salmonella LPS antibody (1:200; Difco) and anti-GM130 monoclonal antibody (1:50; BD Transduction Laboratories) were detected using Alexa Fluor- and Cy5-conjugated secondary antibodies as described above. Nucleic acids were subsequently stained with Hoechst 33342 (2 μg/mL) for 10 min at RT. Intracellular bacteria were then scored for LPS staining. Extruding cells were excluded from analysis because they have a compromised plasma membrane and delivery of antibodies could occur in a digitonin-independent manner. For each experiment, one transwell was used as a permeabilization control to ensure that the apical plasma membrane (but not the endomembranes) was permeabilized. For this, the digitonin-treated cells were incubated with two LAMP1 antibodies: rabbit polyclonal antibody directed against the cytoplasmic tail of LAMP1 (1:250; Novus Biologicals) and a mouse monoclonal antibody directed against the luminal portion of LAMP1 (1:1,000; clone H4A3, Developmental Studies Hybridoma Bank) (Fig. S2). We excluded any experiments in which cells stained with both anti-LAMP1 antibodies.

Immunostaining of mouse gallbladder tissues was performed using previously described procedures (44). Gallbladders were fixed in 4% (wt/vol) PFA for 1 h at RT, washed in PBS, embedded in optimal cutting template compound (Sakura Finetek), and then frozen with isopentane and liquid N2 and stored at −70 °C. Serial sections were cut at a thickness of 4 μm for immunohistochemical staining with Alexa Fluor 488-conjugated rabbit anti-GFP antibody (1:500; Invitrogen) and mouse monoclonal antibody anti-FliC (1:100; BioLegend). Nucleic acids were stained with DAPI.

Animal Infections.

WT S. Typhimurium (Fig. 3 D and E) or S. Typhimurium carrying pMPMA3ΔPlac-PinvF-gfp[LVA] (Fig. 4C) were grown overnight at 37 °C with shaking in LB or LB containing 50 μg/mL carbenicillin, respectively. Cultures were diluted in PBS to ~5–8 × 103 cfu/mL. Female C57BL/6 mice (Charles River) were infected with 100 μL of the diluted culture by tail vein injection. Additionally, for infections with plasmid-bearing Salmonella, mice were treated with carbenicillin (100 mg/kg) by i.p. injection daily throughout the infection to maintain the plasmid. The protocols used were in direct accordance with guidelines drafted by the University of British Columbia's Animal Care Committee and the Canadian Council on the Use of Laboratory Animals. Gallbladders were collected 4–6 d p.i. and processed for EM or immunohistochemistry as described above.

Information on reagents, quantification of cytokine release, fluorescence microscopy, determination of bacterial velocities, and EM is provided in SI Materials and Methods.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Caixia Ma and Tina Huang for their expert technical assistance; Anita Mora for graphics assistance; the Genomics Core Facility at Rocky Mountain Laboratories for DNA sequence analysis; and Rey Carabeo, Ed Miao, Staffan Svärd, and members of the Steele–Mortimer laboratory for discussion and critique of this manuscript. This research was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (O.S.-M. and J.C.) and by grants from the Canadian Institutes of Health Research and the Crohn's and Colitis Foundation (to B.A.V.). M.M. was supported by the Canadian Institute of Gastroenterology/Crohn's and Colitis Foundation of Canada/Canadian Institutes of Health Research Fellowship. B.A.V. is the Canada Research Chair in Pediatric Gastroenterology and the Children with Intestinal and Liver Disorders (CHILD) Foundation Research Scholar.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1006098107/-/DCSupplemental.

References

1. Hybiske K, Stephens RS. Exit strategies of intracellular pathogens. Nat Rev Microbiol. 2008;6:99–110. [PubMed]
2. Wallis TS, et al. The nature and role of mucosal damage in relation to Salmonella typhimurium-induced fluid secretion in the rabbit ileum. J Med Microbiol. 1986;22:39–49. [PubMed]
3. Kent TH, Formal SB, Labrec EH. Salmonella gastroenteritis in rhesus monkeys. Arch Pathol. 1966;82:272–279. [PubMed]
4. Santos RL, et al. Animal models of Salmonella infections: Enteritis versus typhoid fever. Microbes Infect. 2001;3:1335–1344. [PubMed]
5. Vogelsang TM, Bøe J. Temporary and chronic carriers of Salmonella typhi and Salmonella paratyphi B. J Hyg (Lond) 1948;46:252–261. [PMC free article] [PubMed]
6. Buchwald DS, Blaser MJ. A review of human salmonellosis: II. Duration of excretion following infection with nontyphi Salmonella. Rev Infect Dis. 1984;6:345–356. [PubMed]
7. Beuzón CR, Salcedo SP, Holden DW. Growth and killing of a Salmonella enterica serovar Typhimurium sifA mutant strain in the cytosol of different host cell lines. Microbiology. 2002;148:2705–2715. [PubMed]
8. Drecktrah D, Knodler LA, Howe D, Steele-Mortimer O. Salmonella trafficking is defined by continuous dynamic interactions with the endolysosomal system. Traffic. 2007;8:212–225. [PMC free article] [PubMed]
9. Beuzón CR, et al. Salmonella maintains the integrity of its intracellular vacuole through the action of SifA. EMBO J. 2000;19:3235–3249. [PMC free article] [PubMed]
10. Paz I, et al. Galectin-3, a marker for vacuole lysis by invasive pathogens. Cell Microbiol. 2010;12:530–544. [PubMed]
11. Ibarra JA, et al. Induction of Salmonella pathogenicity island 1 under different growth conditions can affect Salmonella-host cell interactions in vitro. Microbiology. 2010;156:1120–1133. [PMC free article] [PubMed]
12. Galán JE, Ginocchio C, Costeas P. Molecular and functional characterization of the Salmonella invasion gene invA: Homology of InvA to members of a new protein family. J Bacteriol. 1992;174:4338–4349. [PMC free article] [PubMed]
13. Van Asten FJ, Hendriks HG, Koninkx JF, Van der Zeijst BA, Gaastra W. Inactivation of the flagellin gene of Salmonella enterica serotype enteritidis strongly reduces invasion into differentiated Caco-2 cells. FEMS Microbiol Lett. 2000;185:175–179. [PubMed]
14. Boddicker JD, Jones BD. Lon protease activity causes down-regulation of Salmonella pathogenicity island 1 invasion gene expression after infection of epithelial cells. Infect Immun. 2004;72:2002–2013. [PMC free article] [PubMed]
15. Drecktrah D, Knodler LA, Ireland R, Steele-Mortimer O. The mechanism of Salmonella entry determines the vacuolar environment and intracellular gene expression. Traffic. 2006;7:39–51. [PubMed]
16. Cirillo DM, Valdivia RH, Monack DM, Falkow S. Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival. Mol Microbiol. 1998;30:175–188. [PubMed]
17. Minamino T, Imae Y, Oosawa F, Kobayashi Y, Oosawa K. Effect of intracellular pH on rotational speed of bacterial flagellar motors. J Bacteriol. 2003;185:1190–1194. [PMC free article] [PubMed]
18. Menendez A, et al. Salmonella infection of gallbladder epithelial cells drives local inflammation and injury in a model of acute typhoid fever. J Infect Dis. 2009;200:1703–1713. [PubMed]
19. Madara JL. Maintenance of the macromolecular barrier at cell extrusion sites in intestinal epithelium: Physiological rearrangement of tight junctions. J Membr Biol. 1990;116:177–184. [PubMed]
20. Mayhew TM, Myklebust R, Whybrow A, Jenkins R. Epithelial integrity, cell death and cell loss in mammalian small intestine. Histol Histopathol. 1999;14:257–267. [PubMed]
21. Rosenblatt J, Raff MC, Cramer LP. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism. Curr Biol. 2001;11:1847–1857. [PubMed]
22. Baron DA, Miller DH. Extrusion of colonic epithelial cells in vitro. J Electron Microsc Tech. 1990;16:15–24. [PubMed]
23. Papaconstantinou HT, et al. Prevention of mucosal atrophy: Role of glutamine and caspases in apoptosis in intestinal epithelial cells. J Gastrointest Surg. 2000;4:416–423. [PubMed]
24. Grossmann J, et al. Induction of apoptosis before shedding of human intestinal epithelial cells. Am J Gastroenterol. 2002;97:1421–1428. [PubMed]
25. Fouquet S, et al. Early loss of E-cadherin from cell-cell contacts is involved in the onset of Anoikis in enterocytes. J Biol Chem. 2004;279:43061–43069. [PubMed]
26. Bullen TF, et al. Characterization of epithelial cell shedding from human small intestine. Lab Invest. 2006;86:1052–1063. [PubMed]
27. Slattum G, McGee KM, Rosenblatt J. P115 RhoGEF and microtubules decide the direction apoptotic cells extrude from an epithelium. J Cell Biol. 2009;186:693–702. [PMC free article] [PubMed]
28. Bergsbaken T, Fink SL, Cookson BT. Pyroptosis: Host cell death and inflammation. Nat Rev Microbiol. 2009;7:99–109. [PMC free article] [PubMed]
29. Foss DL, Zilliox MJ, Murtaugh MP. Bacterially induced activation of interleukin-18 in porcine intestinal mucosa. Vet Immunol Immunopathol. 2001;78:263–277. [PubMed]
30. Monteleone G, et al. Bioactive IL-18 expression is up-regulated in Crohn's disease. J Immunol. 1999;163:143–147. [PubMed]
31. Salvati VM, et al. Interleukin 18 and associated markers of T helper cell type 1 activity in coeliac disease. Gut. 2002;50:186–190. [PMC free article] [PubMed]
32. Wiercinska-Drapalo A, Flisiak R, Jaroszewicz J, Prokopowicz D. Plasma interleukin-18 reflects severity of ulcerative colitis. World J Gastroenterol. 2005;11:605–608. [PubMed]
33. Dupaul-Chicoine J, et al. Control of intestinal homeostasis, colitis, and colitis-associated colorectal cancer by the inflammatory caspases. Immunity. 2010;32:367–378. [PubMed]
34. Zaki MH, et al. The NLRP3 inflammasome protects against loss of epithelial integrity and mortality during experimental colitis. Immunity. 2010;32:379–391. [PMC free article] [PubMed]
35. Cliffe LJ, et al. Accelerated intestinal epithelial cell turnover: A new mechanism of parasite expulsion. Science. 2005;308:1463–1465. [PubMed]
36. Sherman MP, Petrak K. Lactoferrin-enhanced anoikis: A defense against neonatal necrotizing enterocolitis. Med Hypotheses. 2005;65:478–482. [PubMed]
37. Hoiseth SK, Stocker BA. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature. 1981;291:238–239. [PubMed]
38. Knodler LA, et al. Salmonella type III effectors PipB and PipB2 are targeted to detergent-resistant microdomains on internal host cell membranes. Mol Microbiol. 2003;49:685–704. [PubMed]
39. Valdivia RH, Falkow S. Bacterial genetics by flow cytometry: Rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction. Mol Microbiol. 1996;22:367–378. [PubMed]
40. Drecktrah D, et al. Dynamic behavior of Salmonella-induced membrane tubules in epithelial cells. Traffic. 2008;9:2117–2129. [PMC free article] [PubMed]
41. Andersen JB, et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol. 1998;64:2240–2246. [PMC free article] [PubMed]
42. Knodler LA, Bertero M, Yip C, Strynadka NC, Steele-Mortimer O. Structure-based mutagenesis of SigE verifies the importance of hydrophobic and electrostatic residues in type III chaperone function. Mol Microbiol. 2006;62:928–940. [PubMed]
43. Chong A, et al. The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression. Infect Immun. 2008;76:5488–5499. [PMC free article] [PubMed]
44. Khan MA, et al. Toll-like receptor 4 contributes to colitis development but not to host defense during Citrobacter rodentium infection in mice. Infect Immun. 2006;74:2522–2536. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...