Infection with <i>Y</i>. <i>pseudotuberculosis</i> results in an increased frequency and numbers of IFNγ producing NK cells.

<p>Mice were infected with 2 x 10⁷ CFUs of <i>Y</i>. <i>pseudotuberculosis</i> strain YPIII. Three days post infection mLN were excised, single cell suspensions were prepared and used for extracellular (CD3, NK1.1) and intracellular staining (IFNγ). Data from three independent experiments were pooled and analyzed with a Student’s t-test, and the frequency (A) and total number (B) are given (**, p < 0.01; ***, p < 0.001).</p

Global depletion of NK cells lead to increased bacterial titres of <i>Y</i>. <i>pseudotuberculosis</i> in the mLNs.

<p>7-week old female C57BL/6 mice were injected with 100 μg of anti NK1.1 antibody i. p. 24 hours prior to infection. Mice were challenged with 2 x 10⁷ CFU of <i>Y</i>. <i>pseudotuberculosis</i> strain YPIII. (A) Three days post infection mLNs were excised and homogenates were plated onto selective plates. Data from three independent experiments were pooled. Bacterial loads were compared using a Mann-Whitney U test (**, p < 0.01). (B) Three days post infection mLNs were excised and single cell suspensions were stained with Live/Dead, CD3, CD4, CD8, CD19, NK1.1, CD11b, CD11c, CD49b, F4/80, Ly6C, Ly6G. Living cell numbers of macrophages, neutrophils, inflammatory monocytes, B cells, DCs, monocytes, pDCs, TH cells, CTL, NKT-, and NK cells were assessed. Cross striped bars represent undepleted mice, black bars represent NK depleted mice. Data from three independent experiments were pooled and analyzed with a Student’s t-test (**p < 0.01; ***, p < 0.001). DCs: dendritic cells, TH: T helper cells, CTL: cytotoxic lymphocytes, NKT: natural killer T-cells, NK: natural killer cells.</p

TNFα production by macrophages in the mLN is dependent on the presence of NK cells.

<p>24 hours prior to infection mice were injected with 100 μg of anti NK1.1 antibody i.p. mice were infected with 2 x 10⁷ CFUs of <i>Y</i>. <i>pseudotuberculosis</i> YPIII. Three days post infection mLNs were isolated, single cell suspension were prepared and used for extracellular (CD3, CD11b, CD19, CD86, CD49b, MHCII, F4/80) and intracellular staining (TNFα). (A) Depicted are frequencies of CD86⁺/MHCII⁺ macrophages among living cells. CD11b⁺ F4/80⁺ cells were defined as macrophages. Subset analysis with CD86 / MHCII surface markers was performed. (B) Single cells were analyzed for their expression of TNFα and subsequently the expression of either CD3/CD19/CD49b or F4/80 to determine their cell type. Depicted are F4/80⁺ macrophages producing TNFα. Data from two independent experiments were pooled and analyzed with a Student’s t-test (*, p<0.05; ***, p<0.001).</p

<i>Y</i>. <i>pseudotuberculosis</i> YPIII killing by macrophages is increased after IFNγ prestimulation.

<p>1 x 10⁶ macrophages were infected with MOI 10 of YPIII, 1 hour post infection gentamycin treatment killed extracellular bacteria and after additional 60 minutes of incubation cells were lysed and bacteria were plated onto selective plates. The mean of the survival of untreated macrophage was used as baseline. Data from two independent experiments were pooled and analyzed with a Student’s t-test (*, p<0.05).</p

CD11b⁺, CD27⁺ NK cell frequency increases in the mLN during infection with <i>Y</i>. <i>pseudotuberculosis</i> YPIII.

<p>(A) Mice were infected with 2 x 10⁷ CFU of <i>Y</i>. <i>pseudotuberculosis</i> strain YPIII. Three days post infection mLN were excised and NK cell population distribution was analyzed by flow cytometry. To do so, cells were stained with Live/Dead, CD3, NK1.1, CD11b, CD27, CD69 and CD107a. Grey filled squares represent uninfected mice, black filled circles illustrate <i>Y</i>. <i>pseudotuberculosis</i> infected mice. CD11b⁺, CD27⁺ NK cells from the mLN were further analyzed for their expression profiles of the surface markers CD107a (B) and CD69 (C). Grey filled squares represent uninfected mice, while black filled circles represent YPIII infected mice. Data from three independent experiments were pooled and analyzed with a Student’s t-test (*, p < 0.05; ***, p<0.001).</p

Immune response analysis in the PPs of BALB/c mice induced by <i>phoP</i>⁺ and <i>phoP</i>⁻ derivatives of <i>Y. pseudotuberculosis</i> YPIII or IP32953.

<p>Mice were challenged with 2×10⁸ CFU of <i>Y. pseudotuberculosis</i> strains YPIII (<i>phoP</i>⁻) (n = 20), YP149 (YPIII <i>phoP⁺</i>) (n = 20), IP32953 (<i>phoP</i>⁺) (n = 20) and YPIP06 (IP32953 <i>phoP</i>⁻) (n = 20). A control group of uninfected mice (n = 20) was included. At day three postinfection, living cell numbers of B cells and T cells (A), plasmacytoid and conventional DCs (B), NK cells (C), neutrophils (D), monocytes (E) and macrophages (F) in the PPs were analyzed. The data represents the total cell number per organ in a logarithmic scale. Data from four independent experiments were pooled. Statistical analysis was performed using One-way ANOVA with Tukey’s post hoc test (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Immune response analysis in the mesenteric lymph nodes of BALB/c mice infected with <i>phoP</i>⁺ and <i>phoP</i>⁻ derivatives of <i>Y. pseudotuberculosis</i> YPIII or IP32953.

<p>Mice were challenged with 2×10⁸ CFU of <i>Y. pseudotuberculosis</i> strains YPIII (<i>phoP</i>⁻) (n = 20), YP149 (YPIII <i>phoP⁺</i>) (n = 20), IP32953 (<i>phoP</i>⁺) (n = 20) and YPIP06 (IP32953 <i>phoP</i>⁻) (n = 20); a control group of uninfected mice (n = 15) was included. At day three postinfection living cell numbers of B cells and T cells (A), plasmacytoid and conventional DCs (B), NK cells (C), neutrophils (D), monocytes (E) and macrophages (F) in the MLNs were analysed. The data represents the total cell number per organ in a logarithmic scale. Data from four independent experiments were pooled. Living cell numbers were statistically analyzed using One-way ANOVA with Tukey’s post hoc test (*, p<0.05; **, p<0.01; ***, p<0.001).</p

PhoP influences virulence properties of <i>Y. pseudotuberculosis</i>.

<p>BALB/c mice were infected with 5×10⁸ CFU of <i>Y. pseudotuberculosis</i> strains YPIII (<i>phoP</i>⁻) (n = 15), YP149 (YPIII <i>phoP⁺</i>) (n = 15), IP32953 (<i>phoP</i>⁺) (n = 15) and YPIP06 (IP32953 <i>phoP</i>⁻) (n = 15). Survival and fitness of five mice per group were recorded for 14 d. The survival curves were compared with the log-rank test (**, p<0.01).</p

Loss of PhoP in <i>Y. pseudotuberculosis</i> YPIII and IP32953 impairs growth in a low magnesium environment and survival inside J774A.1 macrophages.

<p>(A) <i>Y. pseudotuberculosis</i> strains YPIII (<i>phoP</i>⁻), YP149 (YPIII <i>phoP⁺</i>), IP32953 (<i>phoP</i>⁺) and YPIP06 (IP32953 <i>phoP</i>⁻) were inoculated into TMH medium with either low or high magnesium concentrations (5 mM) and grown at either 25°C or 37°C. The graph represents one independent experiment out of three replicates. Statistical analysis was performed using Two-way ANOVA with Tukey's multiple comparisons test at 24 h of growth (*, p<0.05; ***p<0.001). (B) Activated J774A.1 macrophages were infected with <i>Y. pseudotuberculosis</i> strains YPIII (<i>phoP</i>⁻), YP149 (YPIII <i>phoP⁺</i>), IP32953 (<i>phoP</i>⁺) and YPIP06 (IP32953 <i>phoP</i>⁻) at an MOI 1 for 1 h. The infection was stopped with gentamicin and bacterial uptake was determined 2 h postinfection. Bacterial survival inside the macrophages was determined over time. The graph represents a summary of three independent experiments with four replicates respectively. Statistical analysis was performed using Two-way ANOVA with Tukey's multiple comparisons test at 24 h post assessment of bacterial uptake (***, p<0.001). Error bars represent SEM.</p

PhoP increases expression of <i>cnfY</i> in <i>Y. pseudotuberculosis</i> YPIII.

<p><i>Y. pseudotuberculosis</i> strain YPIII and the isogenic YPIII <i>phoP⁺</i> variant carrying a <i>luxCDABE</i>-reporter plasmid fused to the promoter of <i>cnfY</i> (pJNS02) or the empty vector (pFU54) were grown in LB medium. The <i>luxCDABE</i>-reporter activity and OD₆₀₀ was measured over time. The figure represents three independent experiments done in triplicate. Statistical analysis of the RLU/OD₆₀₀ was performed with the student’s t-test (**, p<0.01; ***, p<0.001).</p