A Precise Temperature-Responsive Bistable Switch Controlling Yersinia Virulence.

Different biomolecules have been identified in bacterial pathogens that sense changes in temperature and trigger expression of virulence programs upon host entry. However, the dynamics and quantitative outcome of this response in individual cells of a population, and how this influences pathogenicity are unknown. Here, we address these questions using a thermosensing virulence regulator of an intestinal pathogen (RovA of Yersinia pseudotuberculosis) as a model. We reveal that this regulator is part of a novel thermoresponsive bistable switch, which leads to high- and low-invasive subpopulations within a narrow temperature range. The temperature range in which bistability is observed is defined by the degradation and synthesis rate of the regulator, and is further adjustable via a nutrient-responsive regulator. The thermoresponsive switch is also characterized by a hysteretic behavior in which activation and deactivation occurred on vastly different time scales. Mathematical modeling accurately mirrored the experimental behavior and predicted that the thermoresponsiveness of this sophisticated bistable switch is mainly determined by the thermo-triggered increase of RovA proteolysis. We further observed RovA ON and OFF subpopulations of Y. pseudotuberculosis in the Peyer's patches and caecum of infected mice, and that changes in the RovA ON/OFF cell ratio reduce tissue colonization and overall virulence. This points to a bet-hedging strategy in which the thermoresponsive bistable switch plays a key role in adapting the bacteria to the fluctuating conditions encountered as they pass through the host's intestinal epithelium and suggests novel strategies for the development of antimicrobial therapies

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

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