Silencing urease: a key evolutionary step that facilitated the adaptation of Yersinia pestis to the flea-borne transmission route

Abstract

The arthropod-borne transmission route of Yersinia pestis, the bacterial agent of plague, is a recent evolutionary adaptation. Yersinia pseudotuberculosis, the closely related food-and water-borne enteric species from which Y. pestis diverged less than 6,400 y ago, exhibits significant oral toxicity to the flea vectors of plague, whereas Y. pestis does not. In this study, we identify the Yersinia urease enzyme as the responsible oral toxin. All Y. pestis strains, including those phylogenetically closest to the Y. pseudotuberculosis progenitor, contain a mutated ureD allele that eliminated urease activity. Restoration of a functional ureD was sufficient to make Y. pestis orally toxic to fleas. Conversely, deletion of the urease operon in Y. pseudotuberculosis rendered it nontoxic. Enzymatic activity was required for toxicity. Because urease-related mortality eliminates 30-40% of infective flea vectors, ureD mutation early in the evolution of Y. pestis was likely subject to strong positive selection because it significantly increased transmission potential.

Keywords: Yersinia urease; arthropod-borne transmission; evolution; plague; pseudogene.

Fig. 1.

The Y. pseudotuberculosis flea toxin is associated with the bacterial membrane. Mortality of fleas 24 h after feeding on blood containing 8.33 mg/mL of a Y. pseudotuberculosis IP32953 whole-cell lysate (CL) or 2.67–5.67 mg/mL proteins of Y. pseudotuberculosis or Y. pestis periplasmic (P), cytoplasmic (C), membrane (M), membrane supernatant (MS), or membrane pellet (MP) fractions. Data are the mean and range from a minimum of three independent experiments. ***P < 0.001, ****P < 0.0001.

Fig. 2.

Urease proteins are present in the toxic subfraction of Y. pseudotuberculosis. (A) Silver-stained 2-DE gels of the membrane supernatant subfraction of Y. pseudotuberculosis and Y. pestis. The numbered spots indicate proteins that were identified as being absent, differentially produced, or with a different pI in Y. pestis compared with Y. pseudotuberculosis profile; boxed numbers indicate urease subunits (Table S1). Molecular mass standards are shown on the left. (B) Organization of the urease cluster in Y. pseudotuberculosis (Y. pstb) IP32953, Y. pestis KIM, and Y. enterocolitica (Y. ent) 8081. The urease locus has a conserved organization in the three species and contains all seven genes that encode the structural (UreABC) and accessory (UreEFGD) proteins of the multimeric urease enzyme. The ureD gene in Y. pestis is a pseudogene (*). The predicted number of amino acids for each protein is noted above the corresponding gene. The positions of primers used for mutagenesis and complementation are indicated by numbered black triangles (identified in Table S2).

Fig. 3.

Yersinia urease is responsible for toxicity to X. cheopis and O. montana fleas. (A) Y. pseudotuberculosis and Y. enterocolitica urease mutants are not toxic to fleas. Mortality of fleas 24 h after feeding on blood containing wild-type, ΔureD, ΔURE, or complemented ΔURE (pWKS-URE_pstb) strains of Y. pseudotuberuclosis IP32953 (Y. pstb) or Y. enterocolitica 8081 (Y. ent). (B) Reactivation of the silenced urease activity in Y. pestis and expression of Y. pseudotuberculosis urease in Y. pestis and E. coli lead to flea toxicity. Mortality of fleas 24 h after feeding on blood containing wild-type Y. pestis KIM6+ with or without the empty cloning vector (WT, pWKS130), KIM6+ expressing the Y. pstb urease cluster (pWKS-URE_pstb), or the Y. pstb ureG-D genes (pCR-ureGD_pstb); KIM6+ in which the ureD pseudogene was repaired (ureDΔG); or E. coli containing the empty cloning vector (pWKS130) or expressing the Y. pstb IP32953 urease operon (pWKS-URE_pstb). Data are the mean ± SD from a minimum of two experiments except for the strain E. coli (pWKS130) for which only one experiment was realized. ***P < 0.001, ****P < 0.0001, ^★the value of both replicates was zero. The result of the in vitro urease test (+ or – phenotype after growth on urea agar) for each bacterial strain is indicated. N/A, not applicable.

Fig. 4.

Toxicity to fleas is dependent on urease enzymatic activity. (A) Mortality of fleas that fed on blood containing Y. pseudotuberculosis cell lysate (CL) or JBU with or without the urease inhibitor p-Bq. Control fleas were fed on blood containing PBS buffer and p-Bq. Data are means ± SD of two independent experiments. (B) Inhibition of urease enzymatic activity by p-Bq in the samples added to the flea blood meals was verified by using a quantitative urease assay. Data are means ± SD of three independent experiments. **P < 0.01. ***P < 0.001, ****P < 0.0001, ^★the value of both replicates was zero.

Fig. 5.

Restoration of urease activity in Y. pestis does not affect infection and blockage rates in fleas that survive the initial acute toxicity. (A) Cumulative blockage rate 4 wk after infection with wild-type Y. pestis KIM6+ or the urease-positive mutant Y. pestis ureDΔG. At least 110 fleas were analyzed per sample. (B) Percentage of fleas infected initially and after 4 wk. (C) Mean bacterial load (cfu) in infected fleas at different times after the infectious blood meal (15–20 fleas per strain and per trial). Data are the mean ± SEM of two trials. NS, nonsignificant difference.