Figure 1.
In vivo expression analysis of the cnfY-luxCDABE fusion.
2×108 bacteria of Y. pseudotuberculosis YPIII pJNS02 (PcnfY::luxCDABE) was used to orally infect BALB/c mice. At indicated time points, mice were anesthesized and bioluminescence was detected with a CCD camera (in vivo imaging system) on the ventral side.
Figure 2.
Expression of PcnfY::gfp in the lymphatic tissues and organs.
2×108 bacteria of Y. pseudotuberculosis YPIII pFU228/pJNS03 (PgapA::dsred, PcnfY::gfpmut3.1) were used to orally infect BALB/c mice. Three days post infection, mice were sacrificed, and PPs, caecum, MLNs, spleen and liver were isolated. Histological slides were prepared and analyzed by fluorescence microscopy to detect bacteria in the tissues by expression of the reporter protein DsRed2. In parallel cnfY expression in the bacteria was monitored by GFPmut3.1 mediated fluorescence. White bars indicate 20 µm.
Figure 3.
Influence of cnfY on the survival of BALB/c mice infected with Y. pseudotuberculosis.
Survival of BALB/c mice (n = 10/strain) were monitored up to 14 days after oral infection with 2×109 cfu of Y. pseudotuberculosis YPIII (black line), the cnfY mutant YP147 (red line) harbouring the empty vector pJNS11, and the strains YPIII pJNS10 (cnfY+) (green line) and YP147 pJNS10 (cnfY+) (blue line).
Figure 4.
Influence of cnfY on the virulence of Y. pseudotuberculosis.
BALB/c mice were infected intragastrically with an inoculum of 2×108 cfu of Y. pseudotuberculosis wild-type YPIII or the cnfY mutant YP147. After 1–7 days of infection, mice were sacrificed and the number of bacteria in homogenized host tissues and organs (PPs, caecum, small intestine, MLNs, spleen, liver) was determined by plating. Data of two independent experiments (5 mice/group) are represented in scatter plots of numbers of CFU per gram as determined by counts of viable bacteria on plates. The statistical significances between the wild-type and the cnfY mutant were determined by the Mann-Whitney test. P-values: *: <0.05; **: <0.01 ***: <0.001.
Figure 5.
Histopathology of H & E stained sections of the ileum and the spleen of mice orally infected with 2×108 bacteria of YPIII and YP147. (A) ileum (6 days post infection); YPIII: diffuse invasion of inflammatory cells into the lamina propria. YP147: Focal invasion of inflammatory cells into the lamina propria; adjacent tissue was unaffected. Bar represents 200 µm. White boxes indicate magnified areas in the slides below. (B) Magnified section of the ileum of an uninfected mouse (left panel), a mouse infected with YPIII - magnification of the ileum selection shown in A (middle panel), and a mouse infected with YP147- magnification of the focal invasion of inflammatory cells (right panel), local inflammation is indicated by the circle. Bar represents 50 µm. (C) spleen (6 days post infection); uninfected mouse (left panel). YPIII infected mouse: splenic atrophy and bacterial colony surrounded by focal necrosis. Pictures shown are representatives of multiple fields and samples of 9 mice. Arrow points to the bacterial foci (middle panel). YP147: hyperplasia of the white pulp and activated lymphoid follicle (right panel). W: white pulp indicated by dashed lines, N: necrosis, H: hyperplasia, R: red pulp, M: muscularis mucosa. Bar represents 50 µm.
Figure 6.
Analysis of immune cells recruited to the spleen after infection with Y. pseudotuberculosis YPIII or YP147.
About 2×108 bacteria (YPIII, YP147) were used to infect BALB/c mice. Three days after infection, mice were sacrificed, the spleens were isolated, homogenized and the cell suspensions were used for flow cytometric analysis. Values on the y axis indicate the numbers of cells isolated from spleen infected with the wild-type strain YPIII or the cnfY mutant strain YP147. CD11b+/Ly6G−: macrophages/monocytes; CD11b+/Ly6G+: neutrophils; CD11c+: DCs; NKp46+: NK cells, CD3+: T cells, CD19+: B cells. The data show the median from at least two different experiments each done with groups of 4–6 mice. The asterisks indicates that there was a significant difference in the number of the indicated cells type in the whole organ in YP147-infected compared to YIPIII-infected mice based on a Mann-Whitney test with * (P<0.05), ** (P<0.01) and *** (P<0.001).
Figure 7.
CNFY-mediated activation of Rho GTPases and morphologic modification and F-actin rearrangements of murine macrophages.
(A) J774A.1 macrophages were treated with 25 nM purified CNFY toxin for 3 h. Toxin treated cells were lysed and aliquots of the cell lysates were taken for westernblot analysis. Remaining samples were incubated with beads-coupled with the GTPase-binding domains of effector proteins interacting with GTP-bound Cdc42, Rac1 and RhoA. The amount of the three different activated Rho GTPases and the total amount of the Rho GTPases of the lysates were analyzed with specific antibodies. (B,C) Fluorescence microscopy of unstimulated (−PMA) and stimulated (+PMA for 48 h) J774A.1 macrophages untreated (B) or treated with 10 nM purified CNFY toxin (C) for 24 h and stained with phalloidin-FITC and DAPI. The arrows indicate actin-rich membrane ruffles, filopodia and stress fibres induced by CNFY incubation.
Figure 8.
CNFY-mediated increase of Yop delivery into epithelial cells and macrophages.
(A) HEp-2 cells were untreated or treated with 25 nM purified CNFY toxin for 3 h prior to the infection with Y. pseudotuberculosis strains YPIII-ETEM (YP173), YP147 ΔcnfY-ETEM (YP217), and YP101 ΔyscS-ETEM (YP174). Cells were labeled with CCF4-AM and analyzed by fluorescence microscopy. Bar: 20 µm (B) HEp-2 cells were untreated or treated with 3 µg/ml (25 nM) purified CNFY toxin for 3 h prior to the infection with Y. pseudotuberculosis strains YPIII-ETEM (YP173) or YP147 ΔcnfY-ETEM (YP217) pregrown at 37°C. (C) Murine macrophages J774A.1 cells were untreated or treated with Rho GTPase modifying toxins CNFY (3 µg/ml), C. botulinum toxin C3 (0.5 µg/ml; 1 µg/ml) and C. difficile toxin TcdBF (85 ng/ml, 250 ng/ml) for 2 h. Subsequently, cell were infected with YPIII-ETEM pregrown at 37°C using a MOI of 10. Cells were labeled with CCF4-AM and percentage of blue HEp-2 cells and macrophages was determined. The graph represents data of two (B) or three (C) independent experiments with 5–6 wells per group. The asterisks indicate the significant difference in the quantity of translocated cells upon infection with the different strains or after toxin treatment based on a Mann-Whitney test with *** (P<0.001).
Figure 9.
Absence of CNFY reduces Yop delivery into neutrophils, macrophages and DCs in PPs, MLNs and spleen during infection.
(A) BALB/c mice were orally infected with 2×109 cfu of YPIII-ETEM (YP173) and YP147 ΔcnfY-ETEM (YP217). YPIII and YP101 ΔyscS-ETEM (YP174) were used as negative controls. Day three post infection the MLNs, liver and spleen were isolated and filtred to generate single-cell suspensions. Cells were labeled with antibodies to the indicated surface markers for macrophages, DCs, neutrophils, NK cells, B and T cells and incubated with CCF4-AM. The percentage of blue cells was analyzed by flow cytometry (see also Fig. S10). Data of 8 mice of which the bacterial burden in the organs has been determined in parallel were normalized to the bacterial loads to determine Yop translocation efficiency. (A) Detection of green and blue cells by flow cytometry in PP, MLNs and spleen of mice infected with YP173 and YP217. The Yop translocation efficiency into all living cells is plotted. (B) The percentage of blue cells of the different analyzed cell types of the MLNs and the spleen were determined and normalized to the bacterial load in the tissues to determine Yop translocation efficiency. The asterisks indicate that Yop translocation efficiency in the different tissues differs significantly between YP147- and YPIII-infected mice based on a Mann-Whitney test with * (P<0.05), ** (P<0.01) and *** (P<0.001).
Figure 10.
Adhesin-mediated interaction of Y. pseudotuberculosis with macrophages induces activation of RhoA which induces T3SS-promoted Yop delivery into the phagocyte. Yop translocation leads to blockage of phagocytosis and YopJ-mediated apoptotic cell death. In CNFY-producing Y. pseudotuberculosis, prior or parallel to the interaction of the bacteria with phagocytes, the CNFY toxin is secreted via outer membrane vesicles. Most likely CNFY-containing vesicles are internalized through receptor-mediated endocytosis, transferred via early endosomes (EE) to acidic compartments (late endosomes, LE) where the toxin is translocated across the bilayer into the cytosol by a low pH-dependent mechanism. Liberation of CNFY leads to activation of small Rho GTPases, in particular RhoA, and this amplifies Yop delivery and cell death. Rho GTPase activation was shown to promote pore formation of the T3SS, which appear to activate caspase-1 [26], [74]. Furthermore, enhanced cell death can lead to an accelerated proinflammatory immune response known to raise the level of activated macrophages in which Yersinia triggers activation of caspase-1 [66]. Induction of caspase-1 leads to secretion of inflammatory cytokines and triggers cell death by inflammatory pyroptosis. In parallel, uncontrolled proliferation of the bacteria will lead to higher toxin concentrations, which will further enhance activation of Rac1, Cdc42 and RhoA and potentiate inflammatory pyroptosis.
Table 1.
Bacterial strains and plasmids.