The host transcriptome during persistent <i>Yersinia</i> infection in the presence and absence of the CNF_Y toxin.

<p>(<b>A</b>) Venn-diagram of differential expressed genes (cut off: Ilog₂FCI≥1.5) from uninfected at 42 dpi versus YPIII or YP147(Δ<i>cnfY</i>) at 42 dpi. (<b>B</b>) Heat map of top enriched (red) and depleted (blue) host transcripts based on <i>DESeq</i>2 analyses. Color-coding is based on <i>rlog</i> transformed read count values. (<b>C</b>) Heat map illustrates log2 fold changes of host transcripts detected in YPIII- or YP147(Δ<i>cnfY</i>)-infected mice compared to uninfected mice (adjusted P value ≤ 0.05). Grey boxes: not significant.</p

Model of CNF_Y influence on the development of <i>Yersinia</i> persistence.

<p>Schematic overview of induced inflammatory and acute phase responses, which are triggered by the <i>Y</i>. <i>pseudotuberculosis</i> strain YPIII and the isogenic CNF_Y-negative variant based on the transcriptome analysis of <i>Yersinia</i>-infected cecal tissue 5 dpi. Host responses that are expected to result from altered inflammatory responses and other defense reactions, are indicated by dashed arrows. Transcript-based adaptations of <i>Y</i>. <i>pseudotuberculosis</i> and the host during colonization of the cecum during acute and persistent infection and their influence on the outcome of the infection are illustrated.</p

Expression pattern of <i>Yersinia</i> persistence genes.

<p>(<b>A</b>) Relative changes in transcript abundance of selected <i>Yersinia</i> genes in YPIII- or YP147 (Δ<i>cnfY</i>)-infected ceca 5 dpi. qRT-PCR was performed with total RNA from Tissue RNA-seq samples. (<b>B</b>) Relative expression of <i>cnfY</i> from total RNA from YPIII- or YP147 (Δ<i>cnfY</i>) grown <i>in vitro</i> at 25°C and 37°C, and isolated from infected ceca 5 and 42 dpi. The data show the mean +/- SEM of at least three independent experiments performed with at least two technical replicates and were analyzed by multiple t-tests employing Holm-Šídák’s correction, P-value: * <0.05, ** <0.01.</p

Cytokine responses in wildtype- and Δ<i>cnfY</i> mutant-infected cecal tissue.

<p>BALB/c mice were orally infected with 10⁶ CFU of YPIII or YP147(Δ<i>cnfY</i>). At indicated time points post infection, the cytokines in the cecal tissue were determined. The bars represent the geometric mean of three independent experiments using n = 3–9 mice/group and the dotted line illustrates the detection limits. The cytokine level at any given time point between wildtype- and Δ<i>cnfY</i> mutant-infected mice was analyzed with the Kruskal-Wallis test and Dunn's correction, P-values: * <0.05, ** <0.01, *** <0.001.</p

Gut microbiota in wildtype- and Δ<i>cnfY</i> mutant-infected mice.

<p>At indicated time points prior (-1) and post infection, feces was sampled from individual mice and tested for <i>Y</i>. <i>pseudotuberculosis</i>. The microbiota composition of persistently <i>Yersinia</i>-infected mice was analyzed by 16S rRNA gene sequencing. (<b>A</b>) Principal coordinates analysis (PCoA) was used to visualize β diversity globally and the bar plot displays the contribution of variables to the observed variance over all time points. (<b>B</b>) Analysis of α diversity using Chao1 index. (<b>C</b>) Relative abundance of the families <i>Desulfovibrionaceae</i> at indicated time points. Relative abundance of the bacteria grouped taxonomically by phyla (<b>D</b>) or microbial orders (<b>E</b>) from 5–6 mice.</p

Tissue alterations during acute and persistent infection of the cecum.

<p>H&E stained sections of the cecal lamina propria (<b>A</b>) and the cecal lymphoid tissue (<b>B</b>) of BALB/c mice at 3 or 42 dpi with about 10⁵⁻10⁶ CFUs of YPIII or YP147(Δ<i>cnfY</i>)<i>/</i>g tissue, or uninfected mice. Cecal lamina propria (3 dpi); YPIII: focal invasion of lymphocytes into the lamina propria (dashed halo) and edema formation (Ed). YP147(Δ<i>cnfY</i>): diffuse distributed granulocytes. E: epithelial cells. Cecal lamina propria (42 dpi); YPIII and YP147(Δ<i>cnfY</i>): isolated granulocytes at the basal lamina propria (dashed halo). Cecal lymphoid tissue (3 dpi); YPIII: massive necrosis, destroyed follicles (dashed halo), ulcus formation (U) and bacterial microcolonies (B) surrounded by invaded granulocytes (black asterisks). E: epithelial cells. YP147(Δ<i>cnfY</i>): necrotic parts and reduced lymphocytes in follicle (dashed halo). Infiltrating granulocytes surround bacterial microcolonies (black asterisks). Pictures show representatives of multiple fields of sections from groups of 3–5 mice. The brackets illustrate the length of the microvilli of the uninfected mice during the acute infection phase. Bar: (<b>A</b>) 50 μm, lower panel, 100 μm upper panel, (<b>B</b>) 100 μm.</p

Precisely adjusted heterogeneous RovA expression is crucial for virulence.

<p>(<b><i>A</i></b>) Fluorescence microscopy of cryosections allows detection of bacteria by expression of the constitutive P_<i>tet</i>-<i>mCherry</i> reporter (mCherry) and revealed heterogeneous expression of the P_<i>rovA</i>-<i>egfp</i>_<i>LVA</i> reporter (eGFP_LVA) in the caecum 3 days post infection. (<b><i>B</i></b>) Quantification of eGFP_LVA-positive cells of the wild-type (YPIII) and the isogenic mutant (YP287) expressing the more stable RovA_{P98S/SG127/128IK/G116A} variant. The mean percentage of RovA-expressing bacteria was significantly higher in the mutant (***, <i>P</i> < 0.001; two-tailed Student’s <i>t</i>-test; <i>n</i> = 40 for each genotype). (<b><i>C</i></b>) Survival of mice infected with <i>Y</i>. <i>pseudotuberculosis</i> revealed reduced virulence of mutants lacking RovA or producing more stable RovA derivatives (**, <i>P</i> < 0.01, ***, <i>P</i> < 0.001; log-rank (Mantel-Cox) test; <i>n</i> = 17 for each genotype). (<b><i>D</i></b>) Infection of mice with 2 x 10⁸ bacteria either deficient in RovA or producing a more stable RovA variant led to reduced colonization of MLNs 3 days post infection (**, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; two-tailed Mann-Whitney test; <i>n</i> = 10 for each genotype). (<b><i>E</i></b>) Model of bistable <i>rovA</i> expression during <i>Y</i>. <i>pseudotuberculosis</i> infection. Upon uptake from the environment, the bacteria express RovA and RovA-induced invasin, which mediates internalization into M-cells. After transcytosis into the subepithelial lymphatic tissues (Peyer’s Patches), most bacteria have switched off <i>rovA</i> expression, thereby limiting their recognition by innate immune cells, but a small RovA ON subpopulation is still found within tissue lesions. During on-going infections heterogeneity could be advantageous for persistence in the caecum as well as reinfection and spreading to other hosts when bacteria are expelled into the intestinal lumen after tissue damage.</p

Identification of a temperature-responsive bistable switch.

<p>(<b><i>A</i></b>) The temperature-responsive <i>Yersinia</i> virulence regulator RovA is autoregulated through positive and negative feedback loops. At 25°C RovA is active and binds cooperatively to a high-affinity site upstream of P2 and activates <i>rovA</i> and <i>invA</i> transcription. When the RovA amount has reached a certain threshold, RovA binds to a low affinity site downstream of P1 to prevent uncontrolled <i>rovA</i> induction. An upshift to 37°C induces a reversible conformational change in RovA that leads to a strong reduction of its DNA-binding capacity and renders this regulator susceptible to proteolysis by the Lon protease. <i>rovA</i> transcription is further regulated by the nutrient-responsive repressor RovM. (<b><i>B</i></b>) <i>Y</i>. <i>pseudotuberculosis</i> wild-type carrying a P_<i>rovA</i>-<i>egfp</i>_<i>LVA</i> fusion was grown at different temperatures and analysed by fluorescence microscopy, and (<b><i>C</i></b>), flow cytometry (one representative replicate; 10⁵ cells). (<b><i>D</i></b>) The percentage of P_<i>rovA</i>-<i>egfp</i>_<i>LVA</i>-expressing wild-type cells quantified by flow cytometry (mean ± SEM; <i>n</i> = 3 for each temperature; 10⁵ cells per replicate), eGFP_LVA-positive cells (ON) are shown in green. The response of P_<i>rovA</i>-<i>egfp</i>_<i>LVA</i> to temperature corresponds to the average RovA level as determined by western blot. Relative RovA amounts were quantified and normalised to the highest temperature for which a homogenous RovA ON population was observed (mean ± SEM; <i>n</i> = 3 for each temperature). (<b><i>E</i></b>), Live cell imaging of <i>Y</i>. <i>pseudotuberculosis</i> expressing P_<i>rovA</i>-<i>egfp</i>_<i>LVA</i> at 32°C. Time series of individual bacteria starting from the OFF state demonstrates switching to the ON and back to the OFF state; representative overlays of eGFP_LVA and bright field images at different time points are shown (also see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006091#ppat.1006091.s013" target="_blank"><b>S</b>1 Video</a>).</p

Thermal shift experiments reveal hysteresis of the temperature-responsive bistable switch.

<p><i>Y</i>. <i>pseudotuberculosis</i> expressing P_<i>rovA</i>-<i>egfp</i>_<i>LVA</i> was grown to a continuous culture in a chemostat at 25°C, shifted for 8 h to 37°C and back to 25°C for 18 h. Bacteria were analyzed by (<b><i>A</i></b>) flow cytometry (mean ± SEM; <i>n</i> = 3 for each temperature; 10⁵ cells per replicate), or (<b><i>B</i></b>) by western blot. Relative RovA amounts were quantified using ImageJ (mean ± SEM; <i>n</i> = 3 for each temperature; c: a protein band unspecifically recognized by the antiserum served as loading control).</p