The Salmonella SPI2 effector SseI mediates long-term systemic infection by modulating host cell migration

Abstract

Host-adapted strains of Salmonella enterica cause systemic infections and have the ability to persist systemically for long periods of time despite the presence of a robust immune response. Chronically infected hosts are asymptomatic and transmit disease to naïve hosts via fecal shedding of bacteria, thereby serving as a critical reservoir for disease. We show that the bacterial effector protein SseI (also called SrfH), which is translocated into host cells by the Salmonella Pathogenicity Island 2 (SPI2) type III secretion system (T3SS), is required for Salmonella typhimurium to maintain a long-term chronic systemic infection in mice. SseI inhibits normal cell migration of primary macrophages and dendritic cells (DC) in vitro, and such inhibition requires the host factor IQ motif containing GTPase activating protein 1 (IQGAP1), an important regulator of cell migration. SseI binds directly to IQGAP1 and co-localizes with this factor at the cell periphery. The C-terminal domain of SseI is similar to PMT/ToxA, a bacterial toxin that contains a cysteine residue (C1165) that is critical for activity. Mutation of the corresponding residue in SseI (C178A) eliminates SseI function in vitro and in vivo, but not binding to IQGAP1. In addition, infection with wild-type (WT) S. typhimurium suppressed DC migration to the spleen in vivo in an SseI-dependent manner. Correspondingly, examination of spleens from mice infected with WT S. typhimurium revealed fewer DC and CD4(+) T lymphocytes compared to mice infected with Delta sseI S. typhimurium. Taken together, our results demonstrate that SseI inhibits normal host cell migration, which ultimately counteracts the ability of the host to clear systemic bacteria.

Figure 1. SseI is required to establish a long-term systemic S. typhimurium infection in mice.

A–C) Mice were infected by IP with WT (circles) or ΔsseI (triangles) strains (6.8×10³ cfu or 6.6×10³ cfu for WT or ΔsseI, respectively), and the cfu from the Peyer's Patches (A), spleen (B), and liver (C) are reported. Only positive error bars are shown for Peyer's patches due to their magnitude. Groups of 5 to 8 mice were analyzed/time point, which were 3, 15, 30 and 45d post-infection (p.i.). The experiment was repeated twice. D–E) Mice were infected by IP with WT(pACYC184) (7.3×10³ cfu), ΔsseI(pACYC184) (6.0×10³ cfu), ΔsseI(psseI) (5.4×10³ cfu, filled triangles), ΔcsgDEFG(pACYC184) (5.4×10³ cfu, squares) bacterial strains. The cfu from the spleen and liver were determined at 45d p.i. (3–5 mice were analyzed per group). Plasmid retention by 45d p.i. was 29.3±7.0% in the spleen (D) and 22.0±7.0% in the liver (E). F) Mice were orally infected with WT (1.5×10⁸ cfu) or ΔsseI (2.06×10⁸ cfu) bacterial strains, and the cfu recovered from Peyer's patches (PP), spleen (Sp), liver, (L), and cecum (C) was measured 34d p.i. (cfu per total PPs shown). The experiment was repeated 3 times; data from a representative experiment is shown. Groups of 5 to 6 mice were analyzed per bacterial strain. *, p<0.05; **, p<0.01, Mann-Whitney U test.

Figure 2. SseI binds directly to the cell migration regulator IQGAP1.

A) Purified GST-SseI (or GSH-resin alone) was used to co-precipitate SseI binding proteins from whole cell extracts of BMDM. Bound proteins were eluted and subjected to SDS-PAGE (4–20% gradient gel) and stained with Coomassie blue. Arrows denote bands that were excised and analyzed by mass spectrometry (top doublet is IQGAP1). Long arrow: non-SseI-specific binding; arrowheads: GST-SseI breakdown fragments. B) GST-SseI (or GST alone) pre-bound to GSH-resin was added to whole cell extracts of BMDM (Mac), activated BMDM or BMDC (DC), and bound proteins were immunoblotted for IQGAP1 (+ = HeLa cell extract). C) Purified His-SseI (WT) or His-SseIC178A (CA) proteins were co-precipitated with IQGAP1-GST or GST alone using GSH-resin and bound SseI was detected by immunoblot using anti-His tag antibody. D) In addition, free IQGAP1 was co-precipitated with His-SseI, His-PipB, or resin prepared from E. coli BL21 extract alone, and IQGAP1 was detected by immunoblot using anti-IQGAP1 antibody. E) BMDM were infected with S. typhimurium-expressing SseI-cya (or SseI alone), and 6h later proteins were immunoprecipitated with anti-CyaA antibody. Bound proteins were immunoblotted for IQGAP1 or TRIP6 (+control = whole cell extract of NIH3T3 cells).

Figure 3. SseI co-localizes with IQGAP1 and actin at the cell periphery.

BMDM were transiently transfected with psseI-EGFP (A) or pEGFP (B) and then fixed and stained for IQGAP1 (red) and actin (phalloidin, blue). Transfected cells were imaged by confocal microscopy (600×), and the white bars represent 17 microns. Arrows indicate regions of co-localization.

Figure 4. SseI causes S. typhimurium-infected BMDM to reverse their direction of travel more frequently.

A) BMDM were seeded onto two-chamber glass slides and infected with GFP-expressing strains of WT (WT(pFPV25.1)) or ΔsseI (ΔsseI(pFPV25.1)) S. typhimurium for 24h. Four locations from each chamber were imaged by time-lapse microscopy (DIC and fluorescence; images were taken every 3min; 45 images were taken in all per movie). The number of times a cell changed its direction of movement more than 90° (per video) are reported for infected cells and their uninfected neighbors (bars represent the median, the data are compiled from 28 total movies (14 movies per bacterial strain) performed in 4 independent experiments, n = 66 for uninfected cells (circles) and n = 82 for infected cells for WT S. typhimurium-infected BMDM (filled circles), n = 75 for uninfected cells (triangles) and n = 96 for infected cells for ΔsseI S. typhimurium-infected BMDM (filled triangles)). **, p-value<0.01 and ***, p-value<0.001; Mann-Whitney U test. B–C) The frames and cell-tracks of two representative movies are shown (B, WT; C, ΔsseI, tracks of uninfected cells are shown in blue and those of infected cells are orange), and the full videos are available online (Videos S1 and S2).

Figure 5. SseI inhibits directed migration of BMDM and BMDC in an IQGAP1-dependent manner.

A) BMDM seeded on transwell filters were infected with the indicated strain of S. typhimurium, and at 24h, heat-killed Salmonella (equivalent of 0 or 12.5 million cfu) was added to the baso-lateral compartment as an attractant. The percentage of cells migrating through the filter was measured 5h later by confocal microscopy. The % migration was calculated: (the % migration of BMDM receiving the heat-killed Salmonella) – (% BMDM migration without the attractant) = % directed BMDM migration. The results are presented as the average of 5 independent experiments; *, p<0.05 and **, p<0.01 when comparing in a two-sample Student's t tests. B) BMDC were seeded and infected as in (A), and then 100ng/ml CCL-19 was used as the chemoattractant to measure the % directed migration (calculated as in (A)). The mean and SEM were calculated from at least 3 replicates. The data are representative of two independent experiments; *, p<0.05 when comparing the % directed BMDC migration to zero in a one-sample Student's t test. C) BMDM from age-matched WT and IQGAP1^−/− mice were treated as in (A) and the results are the average of three independent experiments (*, p<0.05 and **, p<0.01 when comparing in a two-sample Student's t test). D) The amount of WT S. typhimurium protected from gentamicin in WT and IQGAP1^−/− BMDM was measured 2h and 24h after infection and is reported as the average of the total cfu per well; *, p<0.05 when comparing WT to IQGAP1^−/− in a two-sample Student's t test. There was no significant difference in the amount of bacteria protected from gentimicin in BMDM when comparing WT, ΔsseI, and ΔsseI(psseI) S. typhimurium strains (Fig. S1 and data not shown).

Figure 6. In RAW264.7 cells, S. typhimurium mediates SseI-dependent detachment, but SseI does not bind IQGAP1.

A) As in Figure 2B, GST or GST-SseI was combined with whole cell extracts of either WT BMDM or RAW264.7 cells and co-precipitated with GSH-resin. Bound proteins, as well as the indicated amounts of the original whole cell extracts, were subjected to SDS-PAGE and immunoblot detection of IQGAP1. B) WT BMDM and RAW264.7 cells in 6-well plates (5×10⁵ cells/well) were infected with the indicated S. typhimurium strain. After infection, media with or without heat-killed Salmonella was added to the cells, and 24h later, the cells that had detached were harvested, lysed and plated for cfu. The data are presented as the difference between the cfu recovered from cells treated with heat-killed Salmonella and that of untreated cells. The data are representative of 3 independent experiments (each in triplicate); *, p<0.05 in a two-sample Student's t test when comparing WT-infected cells to that of ΔsseI. C–D) BMDM and RAW264.7 cells were seeded onto coverslips and then treated as in part B. The cells were then fixed and stained for S. typhimurium and actin (phalloidin). The % of cells infected (C, 3h and 24h p.i.) and the number of bacteria per infected cell (D, 24h p.i.) were quantified by confocal microscopy. Ten fields (averaging 40 cells/field) were counted per sample group; bars represent the geometric means. ***, p<0.001 when comparing in a two-sample Student's t test.

Figure 7. Cysteine 178 is critical for SseI function in vivo.

A) Amino acid sequences with similarity to the C-terminal domain of SseI (159–244) are shown. Conserved residues are highlighted and C178 of SseI is starred. B) Mice were infected (IP) with equal amounts (4×10³ cfu) of WT and ΔsseI transformed with psseI, psseIC178A, or pACYC184 (empty vector), and the competitive index was measured 2 weeks p.i.. Groups of 4 to 5 mice were analyzed per condition: *, p<0.05 in the Mann-Whitney U test. C) WT and ΔssaV (SPI2 mutant) strains were transformed with psseI-cya or psseIC178A-cya and used to infect RAW264.7 macrophages; the resulting adenylate cyclase activity in macrophage cytosolic fractions was measured at 6h p.i. and is expressed as pmol cAMP/µg protein. *, p<0.05 when comparing WT and ΔssaV in a two-sample Student's t test, and the data are presented as the average of three independent experiments.

Figure 8. SseI-dependent suppression of DC migration in vivo correlates with lower numbers of DC and CD4⁺ T cells in the spleen of mice infected with WT S. typhimurium.

A) BMDC stained with the vital dye PKH26 and infected with GFP-expressing strains of WT (WT(pFPV25.1) or ΔsseI (ΔsseI(pFPV25.1)) S. typhimurium (chased with 100µg/ml gentamicin to kill remaining extracellular bacteria) were injected into 129x1/sv J mice at 5 million cells per mouse. Single cell suspensions were prepared from spleens and analyzed by FACS to detect PKH26 and GFP signals in BMDC that had migrated to the spleen at 6h post-injection. The PKH26-labeld BMDC that were infected with GFP⁺ S. typhimurium ex vivo were also analyzed by FACS to determine input values. The results are expressed as the in vivo migration index = (#PKH26⁺GFP⁺ cells/ #PKH26⁺GFP⁻ cells)_output/(#PKH26⁺GFP⁺ cells/ #PKH26⁺GFP⁻ cells)_input. *, p<0.05; Mann-Whitney U test. B–D) The bacterial loads (B) and the cellular composition (C–D) of the spleens of 129x1/sv J mice infected (IP) with WT, ΔsseI or ΔsseI(psseI) S. typhimurium strains was analyzed at12d p.i. by plating for cfu (B) or by FACS (C–D) for the numbers of DC (C) and CD4⁺ T cells (D) per spleen (Mock = uninfected). *, p<0.05; Mann-Whitney U test. The mean and SEM were calculated from 3–5 replicates per sample. Data from a representative experiment are shown. The experiment was repeated 3 times with similar results.