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.

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 the tight junction marker ZO-1 and the late endosomal/lysosomal marker LAMP1. The percentage of infected cells containing >50 bacteria per cell was scored by fluorescence microscopy (mean ± SD, n ≥ 3 independent experiments). (B) Confocal image showing the two different populations of replicating Salmonella at 8 h p.i. Many hyperreplicating bacteria are not in a LAMP1-positive vacuole. The overlay shows mCherry Salmonella in green, LAMP1 in red, and ZO-1 in blue. (Scale bar, 10 μm; Inset scale bar, 2 μm.) (C) Confocal image showing that some hyperreplicating bacteria are accessible to anti-LPS antibody delivered to the cytosol. At 8 h p.i., the plasma membrane of infected monolayers was selectively permeabilized with digitonin. Cells were then incubated with anti-Salmonella LPS and anti-GM130 antibodies to detect cytosolic bacteria and the cytosolic face of the Golgi, respectively. Nuclei were stained with Hoechst 33342. The overlay shows mCherry Salmonella in red, cytosolic LPS in green, and nuclei in blue. GM130 is shown in the gray scale. Asterisks in the overlay indicate digitonin-permeabilized cells. (Scale bar, 10 μm; GM130 scale bar, 10 μm; Inset scale bar, 2 μm.)

Fig. 2.

Flagella and the invasion-associated T3SS1 are expressed by cytosolic bacteria late during infection. (A–D) Polarized C2BBe1 monolayers were infected with WT Salmonella carrying a plasmid that expresses destabilized GFP [GFP(LVA)] under the control of the T3SS1-associated prgH promoter. At 8 h p.i., monolayers were fixed and immunostained for confocal microscopy. (A) T3SS1 is induced late during infection in “hyperreplicating” bacteria. PprgH-induced bacteria (green in overlay), Salmonella LPS (red), and the tight junction marker ZO-1 (blue) are shown. (Scale bars, 10 μm.) (B) T3SS1-induced bacteria are not in a mature LAMP1-positive SCV. PprgH-induced bacteria (green in overlay), LAMP1 (red), and ZO-1 (blue) are shown. (Scale bar, 10 μm; Inset scale bar, 2 μm.) (C) T3SS1-induced bacteria are cytosolic. The plasma membrane of polarized cells was selectively permeabilized with digitonin before the cytosolic delivery of anti-LPS and anti-GM130 (permeabilization control) antibodies. PprgH-induced bacteria (green in overlay), cytosolic bacteria (LPS, red), and Hoechst 33342 (blue) are shown. Asterisks indicate cells permeabilized by digitonin. GM130 is shown in the gray scale. (Scale bar, 10 μm; GM130 scale bar, 10 μm; Inset scale bar, 2 μm.) (D) T3SS1-induced bacteria are flagellated. PprgH-induced bacteria (green in overlay), FliC (red), and ZO-1 (blue) are shown. (Scale bar, 10 μm; Inset scale bar, 2 μm.) (E) T3SS1-induced bacteria are cytosolic, whereas T3SS2-induced bacteria are vacuolar. Infected cells were fixed at 8 h p.i. and immunostained for LAMP1 or selectively permeabilized with digitonin and incubated with anti-LPS antibodies to detect cytosolic bacteria. The number of T3SS1-induced (PprgH-GFP[LVA]) and T3SS2-induced (PssaG-GFP[LVA]) bacteria positive for LAMP1 or cytosolic LPS staining was scored by fluorescence microscopy (mean ± SD, n ≥ 3 independent experiments.) (F) Some T3SS1-induced bacteria are motile. The motility of T3SS1-induced (PprgH-GFP[LVA]) and T3SS2-induced (PssaG-GFP[LVA]) bacteria was assessed at 8–9 h p.i. by live cell imaging. (Inset) Average instantaneous velocities (μm/s) for motile PprgH-induced bacteria (n = 3 independent experiments).

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. (i) Extruding cell is bacteria-laden. Microvilli on the apical surface of the underlying monolayer are evident. (Scale bar, 2 μm.) (ii) Inset from i. (Scale bar, 0.5 μm.) (B) Transmission EM (TEM) of an extruded cell. Cells were infected as for A, and samples were fixed and processed for TEM. (i) Extruded cell is filled with bacteria and is adjacent to the apical surface of the monolayer, distinguished by microvilli (MV). (Scale bar, 2 μm.) (ii) Inset from i showing the absence of a vacuolar membrane around Salmonella in extruded cells. N, epithelial cell nucleus. (Scale bar, 0.5 μm.) (C) TEM of an infected C2BBe1 cell that was not extruding. Cells were infected as for A, and samples were fixed and processed for TEM. Salmonella are clearly surrounded by a vacuolar membrane (arrowheads). (Scale bar, 0.5 μm.) (D and E) EM of infected gallbladders. Gallbladders from C57BL/6 mice were collected 6 d p.i. and processed for TEM. Apical extrusion of Salmonella-infected epithelial cells into the lumen (L) is evident. Some bacteria are also free in the lumen. (Scale bars, 2 μm.) (E) Infected cell is undergoing extrusion into the luminal space (L), with an obvious constriction site formed by the junctional complexes of neighboring cells (arrowheads). Microvilli (MV) on the apical surface of the mucosal epithelium are indicated.

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]) or T3SS2 (PssaG-GFP[LVA]). At 10 h p.i., monolayers were fixed and immunostained for ZO-1. DNA was stained with Hoechst 33342. Where indicated (+YVAD), 100 μM Ac-YVAD-CMK was added before, and maintained throughout, the infection. Infected (mCherry bacteria) cells, or cells containing PprgH-positive or PssaG-positive bacteria, showing signs of extrusion were scored by fluorescence microscopy (mean ± SD, n ≥ 4 independent experiments). Asterisks indicate significantly different from infected cells (P < 0.05, ANOVA with Dunnett's post hoc test). (B) Confocal image showing an extruding cell containing T3SS1-induced bacteria. Polarized C2BBe1 monolayers infected with WT Salmonella carrying PprgH-GFP[LVA] were fixed and immunostained at 10 h p.i. DNA was stained with Hoechst 33342. PprgH-induced bacteria (green in overlay), apical plasma membrane marker, villin (red), DNA (cyan), and tight junction marker ZO-3 (gray) are shown. The x–z section is indicated by a dashed line. (Scale bar, 10 μm.) (C) Fluorescence microscopy image showing that epithelial cells containing T3SS1-induced, flagellated bacteria are extruded into the gallbladder lumen (L). C57BL/6 mice were infected with Salmonella carrying a plasmid that expresses destabilized GFP under the control of the T3SS1-associated invF promoter (PinvF-GFP[LVA]). Mice were killed 5–6 d p.i., and gallbladders processed for immunostaining. DNA was stained with DAPI. PinvF-induced bacteria (green in overlay), FliC (red), and DNA (cyan) are shown. The arrowhead indicates an extruded cell containing numerous T3SS1-induced, flagellated bacteria. (Scale bar, 10 μm; Inset scale bar, 2 μm.)

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 (red in overlay). At 9 h p.i., live cells were incubated with the active caspase-1 probe FAM-YVAD-FMK (green). Nuclei were stained with Hoechst 33342 (cyan). (Upper) 3D-rendered view in the x–z plane. (Scale bar, 10 μm.) (B) C2BBe1 cells were infected as in A and incubated with FAM-YVAD-FMK (active caspase-1) or FAM-DEVD-FMK (active caspase-3 and -7) at 9 h p.i. Uninfected or infected extruding cells positive for active caspases were scored by fluorescence microscopy (mean ± SD, n ≥ 3 independent experiments). (C) Confocal image of an extruding cell labeled by a membrane impermeant nucleic acid dye. Polarized monolayers were infected with WT Salmonella constitutively expressing GFP (green in overlay). At 10 h p.i., live cells were incubated with two nucleic acid dyes, the membrane impermeant SYTOX Orange (red in overlay) and the membrane permeant Hoechst 33342 (cyan). The extruding cell contains numerous Salmonella and is positive for both nucleic acid stains, indicating a compromised plasma membrane. The x–z section is indicated by a dashed line. (Scale bar, 10 μm.) (D) Apical release of IL-18 from infected epithelial cells. Polarized monolayers were mock-infected (○) or infected with Salmonella (●). Apical supernatants were collected and assayed for IL-18 concentration by sandwich ELISA (mean ± SD, n ≥ 3 independent experiments). (E) Caspase-1 and caspase-3 activation are required for IL-18 release. Polarized monolayers were mock-infected or infected with WT Salmonella. Where indicated, monolayers were pretreated and incubated for the entire infection with the general caspase inhibitor Z-VAD-FMK (50 μM), the caspase inhibitor negative control Z-FA-FMK (50 μM), the caspase-1 inhibitor Ac-YVAD-CMK (100 μM), or the caspase-3 inhibitor, Ac-DEVD-CMK (50 μM). At 10 h p.i., apical supernatants were collected and IL-18 was assayed by ELISA (mean ± SD, n ≥ 3 independent experiments). Asterisks indicate data significantly different from WT Salmonella infection (P < 0.05, ANOVA with Dunnett's post hoc test).