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. 2024 Apr;43(8):1499-1518.
doi: 10.1038/s44318-024-00076-7. Epub 2024 Mar 25.

Salmonella manipulates macrophage migration via SteC-mediated myosin light chain activation to penetrate the gut-vascular barrier

Yuanji Dai #  1   2 , Min Zhang #  1   2 , Xiaoyu Liu #  1   2 , Ting Sun #  3   4 , Wenqi Qi  2 , Wei Ding  5 , Zhe Chen  6 , Ping Zhang  1   2 , Ruirui Liu  1   2 , Huimin Chen  3 , Siyan Chen  2 , Yuzhen Wang  1   2 , Yingying Yue  1   2 , Nannan Song  1   2 , Weiwei Wang  1   2 , Haihong Jia  1   2 , Zhongrui Ma  2   3 , Cuiling Li  1   2 , Qixin Chen  7 , Bingqing Li  8   9   10   11   12
Affiliations

Salmonella manipulates macrophage migration via SteC-mediated myosin light chain activation to penetrate the gut-vascular barrier

Yuanji Dai et al. EMBO J. 2024 Apr.

Abstract

The intestinal pathogen Salmonella enterica rapidly enters the bloodstream after the invasion of intestinal epithelial cells, but how Salmonella breaks through the gut-vascular barrier is largely unknown. Here, we report that Salmonella enters the bloodstream through intestinal CX3CR1+ macrophages during early infection. Mechanistically, Salmonella induces the migration/invasion properties of macrophages in a manner dependent on host cell actin and on the pathogen effector SteC. SteC recruits host myosin light chain protein Myl12a and phosphorylates its Ser19 and Thr20 residues. Myl12a phosphorylation results in actin rearrangement, and enhanced migration and invasion of macrophages. SteC is able to utilize a wide range of NTPs other than ATP to phosphorylate Myl12a. We further solved the crystal structure of SteC, which suggests an atypical dimerization-mediated catalytic mechanism. Finally, in vivo data show that SteC-mediated cytoskeleton manipulation is crucial for Salmonella breaching the gut vascular barrier and spreading to target organs.

Keywords: Salmonella Effector; Cytoskeleton; Host–Microbe Interaction; Kinase; Macrophage Migration.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1. Intestinal CX3CR1+ macrophages carrying Salmonella enters the bloodstream in the early period of infection.
C57BL/6 mice were injected orogastrically with PBS or 2 × 108 CFU per mouse of S. Typhimurium strain expressing GFP, tissue organs, and blood samples were collected at 30 min or 1 h post-infection for subsequent experiments. (A, B) The small intestinal of infected mice were analyzed through immunofluorescence. Small intestinal (SI) after 0.5 h (A) and 1 h (B) of Salmonella infection were stained with PV-1 (Red), DAPI (Blue), and Salmonella (Green). Scale bars, 10 μm. (C) Statistics of Salmonella distribution in host small intestinal at 0.5 h or 1 h after infection. n = 3 per group. (D) Salmonella primarily colonizes macrophages located in the vicinity of blood vessels. Small intestinal (SI) after 1 h of Salmonella infection were stained with F4/80 (Red), DAPI (Blue), and Salmonella (Green). The white dashed line indicates F4/80-positive macrophages containing bacteria. Scale bars, 10 μm. (E) The number of bacteria recovered from 100 μL peripheral blood per mouse. n = 6 per group. Data were mean ± SD. An unpaired t-test was used to determine the statistical significance between the two groups. ****P < 0.0001. (F, G) The white blood cells were isolated from infected C57BL/6 mice, and the GFP-positve cells were analyzed by flow cytometry 1 h post-infection. n = 6 per group. Data were mean ± SD. An unpaired t-test was used to determine the statistical significance between the two groups. **P < 0.01. (H, I) Representative flow cytometric analysis (H) and percentage (I) of dendritic cells (CD11c+F4/80−) and macrophage cells (F4/80+) in GFP-positve population from white blood cells from six mice per group. Data were mean ± SD. An unpaired t-test was used to determine the statistical significance between the two groups. ***P < 0.001. (J, K) Representative flow cytometric analysis (J) and percentage (K) of intestinal macrophage cells (CX3CR1+F4/80+) in GFP-positve population. n = 6 per group. Data were mean ± SD. An unpaired t-test was used to determine the statistical significance between the two groups. ***P < 0.001. Each dot indicates an individual animal. (L) Representative images of blood cells containing Salmonella (Red) associated with F4/80-expressing macrophage cells (Green), determined by confocal microscopy. Scale bars, 2 μm. Source data are available online for this figure.
Figure 2
Figure 2. Salmonella infection induces the migration and invasion properties of macrophages in an actin- and SteC-dependent manner.
(A, B) The migration and invasion of RAW264.7 cells infected by Salmonella were detected at the indicated MOI for 24 h. The cells that migrated to the lower chamber were stained and counted under light microscopy. Scale bars, 50 μm. The uninfected RAW264.7 cells were used as negative controls. LPS (1 μg/ml) and heat-treated Salmonella (95 °C 10 min, MOI 10:1) were also used as controls. (C) The migration and invasion of RAW264.7 cells treated by CCL2 (10 ng/ml) or CCL7 (100 ng/ml) or challenged by wild-type Salmonella (MOI 10:1) with or without colchicine (50 μM) or Latrunculin B (50 μM). (D) The migration and invasion of RAW264.7 cells were challenged by wild-type, ΔssaW, ΔsteC, or ΔsseI. MOI = 10. (E) The actual movement trajectory of bone marrow-derived macrophages (BMDM) is challenged by wild-type or ΔsteC. The uninfected BMDM cells were used as controls. More than 30 individual cells for each group were calculated. (F) The migration and invasion of BMDM cells infected by wild-type or ΔsteC were detected for 4 h. The uninfected BMDM cells were used as controls. (G) RAW264.7 cells infected by wild-type or ΔsteC expressing GFP (green) were stained for F-actin (red) and DAPI (blue). Scale bar, 2 μm. (H) The mean fluorescence intensity of intracellular F-actin related to H was quantified. More than 20 individual cells for each group were calculated. Data information: For 2B, 2C, 2D, and 2F, the amounts of migration and invasion of uninfected RAW264.7 cells or BMDM cells were set as 1. The mean ± SD of three biological replicates was shown. For 2H, The normalization of actin intensity is determined through the division of fluorescence intensity by the area. An unpaired t-test was used to determine the statistical significance between the two groups. The mean ± SD of 20 individual cells was shown. Unless otherwise specified, the infected group was compared with the uninfected control group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. not significant. Source data are available online for this figure.
Figure 3
Figure 3. SteC promotes macrophage migration and invasion and actin rearrangement in a MEK/MLCK-independent manner.
(A)The expression of SteC were detected in RAW264.7 cells expressing GFP (pGFP), SteC (pSteC), or SteC K256H (pSteC K256H) using western blotting. (B–D) The migration and invasion of pGFP, pSteC, or pSteC K256H. The cells that migrated to the lower chamber were stained and counted under light microscopy. Scale bars, 50 μm. The amounts of migration and invasion of pGFP were set as 1. (E) RAW264.7 cells expressing GFP, SteC, or SteC K256H were stained for F-actin (red) and DAPI (blue). Scale bar, 5 μm. (F) The fluorescence intensity of intracellular F-actin related to (D) was quantified. More than 20 individual cells were measured for each group. (G) Schematic of traditional MLC activation pathway and corresponding inhibitors. PD98059: MEK inhibitor, ML-7: MLCK inhibitor, Y-33075: ROCK inhibitor, Latrunculin B: actin polymerization inhibitor. (H) The migration and invasion of pSteC with or without four inhibitors. The amounts of migration and invasion of pSteC without inhibitors (DMSO) were set as 100%. (I) Four indicated inhibitors were added to pSteC to determine their impact on F-actin rearrangement. F-actin (red), DAPI (blue), GFP (Green). Scale bar, 5 μm. (J) The fluorescence intensity of intracellular F-actin related to (I) was quantified. More than 20 individual cells for each group were measured. Treatments were normalized to DMSO control. Data information: The mean ± SD of three biological replicates was shown. An unpaired t-test was used to determine the statistical significance between the two groups. Unless otherwise specified, the infected group was compared with the uninfected control group. *P < 0.05, ***P < 0.001, ****P < 0.0001, n.s. not significant. Source data are available online for this figure.
Figure 4
Figure 4. SteC directly interacts with Myl12a in vivo and in vitro.
(A) Yeast two-hybrid screening of SteC-interacting proteins of macrophage. SteC was used as the bait to screen a RAW264.7 AD library. The sequencing results showed that fourteen positive clones corresponded to Myl12a. (B) Pairwise verification of the SteC-Myl12a interaction. (C) Representative confocal images show GFP-SteC co-localizes with mCherry-Myl12a. Scale bar, 10 μm. (D) The fluorescence signal intensity on the white dashed line was plotted. (E) Pearson’s colocalization coefficient (PCC) value of SteC-Myl12a colocalization from 20 different cells. The mean ± SD of 20 individual cells was shown. (F) Pull-down assays of His6-Myl12a and SteC FL and SteC-C were performed. SteC FL: full-length SteC; SteC-C: SteC 194 aa–457 aa. (G, H) The binding affinities of SteC FL or SteC-C and Myl12a were measured by BLI. Source data are available online for this figure.
Figure 5
Figure 5. SteC phosphorylates and activates Myl12a in vivo and in vitro.
(A) In vitro phosphorylation assays of Myl12a catalyzed by SteC using γ32P-ATP. The experiments were performed using purified Myl12a and SteC full-length protein or protein fragments. 32P Autorad, autoradiography of 32P-labeled proteins. (B) In vitro phosphorylation assays of Myl12a catalyzed by SteC using Phos-tag gel. P-Myl12a: Phosphorylated Myl12a. (C) Mass spectra of Myl12a and Myl12a phosphorylated by SteC confirm that the molecular weight of SteC-modified Myl12a increased by 160 Da compared to the native Myl12a. (D) MS/MS spectra of phosphorylated peptides of SteC-modified Myl12a. The b and y ions are indicated along the peptide sequence above the spectra. (E) Reaction products of in vitro phosphorylation of Myl12a were immunoblotted with the indicated antibodies. (F) In vitro phosphorylation assays of Myl12a T19AS20A by SteC using Phos-tag gel. P-Myl12a: Phosphorylated Myl12a. (G) The phosphorylation levels of Myl12a during infection were detected using western blotting in RAW264.7 cells 1 h post-WT or ΔsteC infection. (H) The phosphorylation levels of Myl12a during infection were assessed using western blotting in BMDM cells 1 h post-WT or ΔsteC infection. (I) The phosphorylation levels of Myl12a in BMDM was quantified using western blotting at different times after infection with wild-type Salmonella. (J) The phosphorylation level of Myl12a in the indicated cells was quantified using western blotting. (K) The phosphoproteomic data determined by LC-MS/MS was assessed by principle-component analysis (PCA). WT: pGFP, STEC: pSteC. (L) Volcano plot for differentially phosphorylated peptides identified in pSteC and pGFP. Student’s t-test was used to determine the statistical significance between the two groups. The top ten differential proteins in phosphorylation are labeled and Myl12b is highlighted. Source data are available online for this figure.
Figure 6
Figure 6. Structural insights into the catalytic and Myl12a-recruited mechanisms of SteC.
(A) The upper panel depicts a schematic representation of SteC’s domains and fragments. The lower panel depicts the overall structure of SteC predicted by trRosetta server. (B) In vitro phosphorylation assays were performed with SteC full-length and fragments. The samples were analyzed using Phos-tag gel. P-Myl12a: Phosphorylated Myl12a. (C) The structure of SteC-ATP was obtained from molecular dynamic simulation based on SteC-AMP structure (PDBid: 8JBI). The zoomed view of the SteC-ATP interface is marked with a black frame. The essential residues and ATP were shown in stick mode. (D) The proposed mechanism of SteC-mediated phosphorylation. Asp364 acts as the general base for nucleophilic attack. (E) The dimeric structure of SteC is shown in surface mode. ATP molecule bound in the hydrophobic pocket of SteC dimer. (F) Sequence Logo of the multiple sequence alignment in the active sites of SteC. (G) Activity of SteC or active site mutants to phosphate Myl12a. (H) The binding affinities of SteC fragments and Myl12a were measured by BLI. (I) Model of SteC-Myl12a complex. The zoomed view of the SteC-Myl12a interface is marked with a black frame. The residues from the SteC M-helix of the interface are highlighted by a purple circle. (J) The binding affinities of SteC mutants and Myl12a were measured by BLI. Source data are available online for this figure.
Figure 7
Figure 7. SteC is indispensable for gut-vascular barrier breakthroughs and systemic spreading during Salmonella infection.
(A)The proliferation of WT or ΔsteC strains in RAW264.7 cells were measured by counting CFU. The bacterial count after a two-hour invasion was set to 1 for the WT or ΔsteC group, respectively. n = 3 per group. (B) Representative images of intracellular bacteria by electron microscopy. RAW264.7 cells were observed 10 h post-infection with WT or ΔsteC strains. Scale bar, 5 μm. (C–E) C57BL/6 mice were orally infected with WT or ΔsteC for 1 h. (C) Statistics of Salmonella distribution in host small intestinal after infection. n = 5 per group. (D, E) The number of indicated bacteria in the blood (D) or liver (E) 1 h post-infection. n = 3 per group. (F) The small intestinal of infected mice were analyzed through immunofluorescence. BALB/C mice were orally infected with WT or ΔsteC. Small intestinal (SI) blood vessels were stained with PV-1 (Red). DAPI (blue). Salmonella (Green). Scale bars, 10 μm. (G) In vivo bioluminescence imaging of BALB/C-nu mice inoculated intragastrically with WT or ΔsteC expressing luciferase. Representative luminescence images show WT Salmonella spreads to the liver, spleen, and lymph nodes earlier than ΔsteC. (H) Representative images depicting the distribution of WT or ΔsteC in the small intestine after a 20-hour infection. PV-1 (Red). DAPI (blue). Salmonella (Green). Scale bars, 10 μm. (I) Quantification and distribution of Salmonella within small intestinal cells at 20 h post-infection in each experimental group analyzed by 25–30 visual fields of three mice. (J) Schematic diagram illustrating the vascular penetration assay. (K) HUVEC cells were seeded into the transwell chamber and allowed to incubate overnight, followed by fixation with 4% paraformaldehyde. RAW264.7 cells infected by WT or ΔsteC were added and co-cultivated for 24 h. The cells that migrated to the lower chamber were stained and counted under light microscopy. n = 3 per group. The uninfected RAW264.7 cells were used as negative controls. (L) Representative imaging results SteC-expressing RAW264.7 cells penetrating the HUVEC cell barrier. Scale bars, 10 μm. (M) Schematic model of SteC-mediated Salmonella dissemination. Data information: The mean ± SD was shown. An unpaired t-test was used to determine the statistical significance between the two groups. Unless otherwise specified, the infected group was compared with the uninfected control group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. not significant. Source data are available online for this figure.
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