The presence of professional phagocytes dictates the number of host cells targeted for Yop translocation during infection

Abstract

Type III secretion systems deliver effector proteins from Gram-negative bacterial pathogens into host cells, where they disarm host defences, allowing the pathogens to establish infection. Although Yersinia pseudotuberculosis delivers its effector proteins, called Yops, into numerous cell types grown in culture, we show that during infection Y. pseudotuberculosis selectively targets Yops to professional phagocytes in Peyer's patches, mesenteric lymph nodes and spleen, although it colocalizes with B and T cells as well as professional phagocytes. Strikingly, in the absence of neutrophils, the number of cells with translocated Yops was significantly reduced although the bacterial loads were similar, indicating that Y. pseudotuberculosis did not arbitrarily deliver Yops to the available cells. Using isolated splenocytes, selective binding and selective targeting to professional phagocytes when bacteria were limiting was also observed, indicating that tissue architecture was not required for the tropism for professional phagocytes. In isolated splenocytes, YadA and Invasin increased the number of all cells types with translocated Yops, but professional phagocytes were still preferentially translocated with Yops in the absence of these adhesins. Together these results indicate that Y. pseudotuberculosis discriminates among cells it encounters during infection and selectively delivers Yops to phagocytes while refraining from translocation to other cell types.

Fig. 1. Neutrophils, macrophages, and dendritic cells are preferentially translocated with YopH during Yptb infection

Mice were orogastically infected with 2x10⁹ CFU of WT-HTEM. Day 5 post-infection tissues were harvested and filtered to generate a single cell suspension. Cells were incubated with CCF2-AM and labeled with antibodies to the indicated cell surface marker(s). Fluorescence intensity was analyzed by flow cytometry. (A) Detection of green and blue cells by flow cytometry in PP of uninfected mice or mice infected with WT or WT-HTEM. The blue⁺ cells are gated (R1) with the percentage of blue⁺ cells indicated in the gate. (B, D, F) The percentage of blue⁺ cells in the cell type indicated on the x-axis in (B) PP, (D) MLN, and (F) spleen. (C, E, G) The percentage of a cell type present in the entire organ (white bars) and the percentage of specific cell type present in the blue⁺ population (black bars) in the (C) PP, (E) MLN, and (G) spleen. The fold enrichment of each cell type in the blue⁺ population compared to total of that cell type in the whole organ is indicated above each set of bars. The experiment was repeated 4 times and the bars are the average + SEM from all the experiments. The asterisk indicates that there was a significant difference in the number of the indicated cell type in the blue⁺ population compared to that same cell type in the whole organ based on a unpaired, two tailed, t test (P<0.05). Note the Y-axis in (C) differs from that of (E) and (G).

Fig. 2. HTEM and YopE are translocated into the same cells in the spleen during infection

Four mice were infected orogastrically with WT-HTEM and five days post-infection, spleens were harvested, pooled, and labeled with CCF2-AM and antibody to GR1. (A) Gr1+ cells were sorted based on their blue/green fluorescence into blue^neg/green⁺ (R1), blue^low/green⁺ (R2), and blue^high/green⁺ (R3) populations. B. Western blot of 2x10⁵ GR1⁺ cells per gate. The lanes were loaded as follows, R1 blue^neg (uncleaved CCF2-AM); R2 blue^low (some CCF2-AM conversion); R3 blue^high. Blots were probed with antibody to TEM, YopE and actin, which was used as a loading control. The blot is representative of three independent experiments.

Fig. 3. Localization of Yptb in the PP, MLN and spleen

Immunohistochemical analysis of (A–D) PP, (E–G) MLN, and (H–N) spleens after 5 days infection with 2x10⁹ CFU WT-HTEM. Sections were stained with α-Yptb and hemotoxylin to identify Yptb and distinguish B and T cells from areas of inflammation. The location of Yptb was detected by 3,3′-diaminobenzidine which produces brown staining (indicated by white arrows). Black arrows indicate areas of inflammation. (A, E, H) Uninfected tissues (10X); (B–C, F–G, I–J, L–M) areas of inflammation in infected tissues (10X and 60X); (C, G arrow 3, M) colonies detected in areas of infiltration (60X); (D, G arrow 1, N) colonies detected in B and T cell areas (60X); (G arrow 2, L) colonies detected at boundary zone (60X). (O–P) Quantification of microcolonies is areas of inflammation, B and T cell zones, and boundary zones in (O) PP, (P) MLN, and (Q) spleens from 5 mice; 1–2 sections/mouse were analyzed and scored by 3 independent viewers.

Fig. 4. Neutrophil depletion and suppression of inflammation reduces the total amount of translocated Yops

(A–D) Mice were injected intraperitoneally with an isotype control (α-IgG2bκ) or the RB6-85C (α-GR1) rat monoclonal antibodies 1 day prior to and 2 days after oral infection with WT-HTEM. Three days post-infection, mice were sacrificed, the PP harvested and the number of blue⁺ and GR1⁺ cells were quantified. (A) Percent GR1+ cells in PP of an isotype control treated and an RB6-8C5 treated mouse by flow cytometry. The percentage of GR1+ cells (gated in R3) is indicated on the plots. (B) Percent blue⁺ cells from PP (gated in R1) of isotype control treated mice and RB6-8C5 treated mice. (C) The percentage of blue⁺ cells (x-axis) were plotted versus the percentage of GR1⁺ cells (y-axis) in the PP from mice treated with the isotype control antibody (squares) and mice treated with α-GR1 (triangles). The experiment was repeated twice and data from all the mice are shown. Significant differences between both the GR1+ cells and the number of blue⁺ cells from the two groups of mice were determined by an unpaired, two tailed t test. Both the percentage of GR1+ cells and the percentage of blue⁺ cells in each group of mice were statistically different (unpaired, two tailed, t test; * indicates P<0.01) (D) Bacterial colonization in PP of the isotope control or RB6-8C5-treated mice shown as CFU/gm PP. Each dot represents one mouse; the bar represents the geometric mean. There was no statistical difference in the bacterial load between the two populations of mice by an unpaired, two tailed, t test, P=0.54. (E–F) Mice were intragastically infected with 2x10⁹ YPIII-HTEM or YPIIIyopER144A-HTEM and the number of blue⁺ and Gr1⁺ cells were quantified by flow cytometry. 5 days post-infection PP were harvested. (E) The percentage of Gr1⁺CD11b⁺ cells and blue⁺ cells in the PP were plotted from mice infected with YPIII-HTEM (squares) or YPIIIyopER144A (triangles). The experiment was repeated three times and data from all mice are shown. Significant differences between the GR1+ and the blue⁺ cells between the two groups of mice were determined by an unpaired, two tailed t test. (F) Bacterial colonization in mice infected with YPIII or YPIIIyopER144A-HTEM shown as CFU/gm PP. There was no statistical difference in bacterial load by an unpaired two tailed t test P=0.59

Fig. 5. WT-HTEM preferentially targets macrophages, neutrophils and dendritic cells from splenocytes at a low, but not high MOI

Splenocytes were infected with WT-HTEM (A, B) at a MOI of 20:1 or (C, D) at a MOI 1:1, incubated with CCF2-AM, and then distinguished by cell type by antibody and flow cytometry. The number of blue cells of a particular cell type was quantified and the number of each cell type in the spleen was quantified. (A, C) The percentage of blue cells (y axis) in the cell type indicated on the x-axis. (B, D) The percentage of each cell type present in the entire organ (white bars, y-axis on left) compared to the percentage of each cell type present in the blue⁺ population (black bars, y-axis on right). The fold enrichment of each cell type in the blue⁺ population when compared to the same cell type in the organ is denoted on top of each pair of bars. The experiment was repeated 4 times and the average + SEM are plotted. The asterisk indicates that there was a significant difference in the percentage of a specific cell type in the blue⁺ cells compared to the entire organ (unpaired, two tailed t test P<0.05).

Fig. 6. Yptb binds preferentially to professional phagocytes cells at low MOI

(A) A single cell suspension of splenocytes was treated with 2 μM of cytochalasin D and then infected with WT IP2666 expressing GFP at a MOI of 1:1 for 1 hour. Splenocytes were labeled with antibodies to different cell markers and the binding of Yptb to specific cell types was analyzed by ImageStream. The experiment was done twice and the average number of a particular cell type associated with Yptb is shown. (B) A single cell suspension of splenocytes was treated with 2 μM of cytochalasin D (white bars) or left untreated (black bars) and then infected with WT IP2666 expressing GFP at a MOI of 1:1 for 1 hour. Splenocytes were labeled with antibodies to different cell markers and the binding of Yptb to specific cell types was analyzed by flow cytometry. The experiment was repeated 3 times and the average + SEM are graphed. The asterisk indicates significant differences between cytochalasin D treated and untreated cells as determined by t test (P<0.05). (C–D) Cells were treated with 2μM of cytochalasin D and infected with WT, Δinv, or ΔyadA expressing GFP at MOI 1:1 for 1 hour. (C) The percent cells bound to GFP-expressing bacteria was determined by fluorescence intensity in the FITC channel of the total splenocyte population. (D) The percentage of specific cell types in spleens bound by WT, Δinv and ΔyadA. The experiment was repeated 4 times and the average + SEM are graphed. The asterisk indicates significant differences in the association of the indicated cell type by WT versus ΔyadA infected cells as determined by t test (P<0.05).

Fig. 7. ΔyadA and Δinv translocate HTEM into fewer numbers of splenocytes

Splenocytes were left uninfected or infected with WT-HTEM, ΔyopB-HTEM, Δinv-HTEM, or ΔyadA-HTEM at an MOI of 1:1, incubated with CCF2-AM and antibodies to distinguish particular cell types. (A) The percentage of blue⁺ cells was determined by flow cytometry. (B) The relative percent of blue+ cells by setting WT-HTEM to 100% and normalizing the percentage of blue cells of the HTEM mutant strain to WT-HTEM. Experiments with the Δinv-HTEM, ΔyadA-HTEM and ΔinvΔyadA-HTEM were repeated 9, 13 and 6 times, respectively. Differences were determined by using a paired t test with the WT-HTEM sample. (C) The percentage of blue⁺ cells in the indicated cell type after infection with Δinv-HTEM, ΔyadA-HTEM or ΔinvΔyadA-HTEM was compared to infection with WT-HTEM. (D) The percentage of a cell marker in the blue cell population after infection with Δinv-HTEM, ΔyadA-HTEM or Δinv ΔyadA-HTEM was compared to infection with WT-HTEM. (C–D) The bars represent the average + SEM of at least 8, 12 and 6 experiments for Δinv-HTEM, ΔyadA-HTEM and ΔinvΔyadA-HTEM analyzed with the indicated markers. Asterisk indicates significant differences between the number of blue cells in the WT-HTEM infected population versus the adhesin mutant population as determined by paired, t test (P<0.05).

Fig. 8. YadA and Invasin are critical for colonization of Peyer’s patches

Seven to nine week old female BALB/c mice were infected orally with 2x10⁹ CFU of WT-HTEM, ΔyadA-HTEM, Δinv-HTEM or ΔinvΔyadA-HTEM and sacrificed at 3 or 5 days post infection. Each dot represents one mouse; the bars represent the average. (A) At each time point PP were harvested, a cell suspension was generated and plated for CFU. (B) Cells were incubated with CCF2-AM and the percentage of blue cells in PP was determined by flow cytometry at each time point (C) Cells were labeled with α-Gr1-PeCy5 and α-Cd11b-PeCy7 to determine the percentage of neutrophils by flow cytometry. Each dot represents data from an individual mouse. Bars indicate the geometric mean in (A) and average in (B) and (C). The experiment was repeated three-five times and all the data was combined and analyzed using ANOVA with Tukey-Kamer multiple comparison. * indicates P value < 0.01 and ** indicates P value < 0.001. (D) The percentage of blue⁺ cells (x-axis) were plotted versus the percentage of GR1⁺CD11b⁺ cells (y-axis) in the PP from mice colonized with comparable numbers of either WT-HTEM (squares) or Δinv-HTEM (triangles). (E) Bacterial colonization in PP of the WT-HTEM and Δinv-HTEM mice used in panels D, F and G. Each dot represents one mouse; the bar represents the geometric mean. There were no significant differences. (F–G) The percentage of Gr1+CD11b+ (F) or GR1+CD11b− (G) cells in the blue cell population in the PP after infection with either WT or Δinv-HTEM. There were no significant differences.