yadBC of Yersinia pestis, a new virulence determinant for bubonic plague

Abstract

In all Yersinia pestis strains examined, the adhesin/invasin yadA gene is a pseudogene, yet Y. pestis is invasive for epithelial cells. To identify potential surface proteins that are structurally and functionally similar to YadA, we searched the Y. pestis genome for open reading frames with homology to yadA and found three: the bicistronic operon yadBC (YPO1387 and YPO1388 of Y. pestis CO92; y2786 and y2785 of Y. pestis KIM5), which encodes two putative surface proteins, and YPO0902, which lacks a signal sequence and likely is nonfunctional. In this study we characterized yadBC regulation and tested the importance of this operon for Y. pestis adherence, invasion, and virulence. We found that loss of yadBC caused a modest loss of invasiveness for epithelioid cells and a large decrease in virulence for bubonic plague but not for pneumonic plague in mice.

FIG. 1.

Schematic diagram of the yadBC operon, with coordinates for the open reading frames, signal sequences, and antigenic regions used in this study.

FIG. 2.

Growth and yadBC promoter activity from yadBC-lacZ integrated within invA in the chromosome of Y. pestis CO99-3015.S10. Y. pestis CO99-3015.S10 and the negative control strain Y. pestis CO99-3015.S4 with promoterless lacZ integrated into invA were grown at 26 and 37°C, and the OD₆₂₀ and β-galactosidase activity were determined at the indicated times. β-Galactosidase levels (expressed in Miller units) were determined for yersiniae grown at 26°C (▴) and at 37°C (▪); corresponding OD₆₂₀ values are also shown (▵ and □, respectively). The open circles show β-galactosidase activity at 37°C for Y. pestis CO99-3015.S4. Essentially identical data were obtained for this strain grown at 26°C, and the growth data for this strain overlapped those for Y. pestis CO99-3015.S10 (data not shown). The experiments were done three times on different days with similar results. The data shown are data from one experiment, and the error bars indicate the ranges for duplicate samples.

FIG. 3.

Test for possible quorum-sensing effect of HSL molecules present in the culture supernatant on yadBC promoter activity. (A) Promoter activity resulting from single-crossover integration of pSucinv::yadBC-lacZ in Y. pestis strain CO99-3015.S10 in response to fresh medium (negative control) (×) and to cell-free culture supernatants of Y. pestis strains KIM6+ (positive control supernatant) (□), CO99-3015.S1 (▵), and KIM6-2109+ (“quorum-sensing triple mutant,” negative control supernatant) (⧫). The data are averages ± standard deviations of triplicate assays. (B) Promoter responsiveness of the E. coli MG4(pKDT17) reporter strain (positive control). The medium and symbols are the same as those described above for panel A.

FIG. 4.

Expression of pBAD24YadBC in a ΔyadBC Y. pestis strain. Y. pestis CO92.S13(pBAD24YadBC) and Y. pestis CO92.S13(pBAD24) (vector control) were grown at 37°C, 0.25% arabinose was added, and incubation was continued for 4 h to induce expression of YadB and YadC. Whole cells were solubilized and analyzed by using immunoblots probed with anti-YadB (right panel) or anti-YadC (left panel) antibodies. Lane BC⁺⁺, Y. pestis CO92.S13(pBAD24YadBC); lane V, vector control [Y. pestis CO92.S13(pBAD24)]. The positions of molecular mass markers are indicated on the left. Open arrows indicate bands thought to represent monomeric forms of YadB and YadC; putative trimeric forms are indicated by solid arrows; and the arrowheads indicate oligomeric forms that react with both anti-YadB and anti-YadC antibodies.

FIG. 5.

Invasiveness of the parent strain of Y. pestis CO92.S1 and the yadBC deletion mutant strain CO92.S8 with WI-26 and HeLa epithelioid cell lines. Invasion was measured by performing gentamicin protection assays in triplicate, and the data are averages and standard deviations. The three independent WI-26 experiments and the two HeLa experiments were positive for significance as determined by the sign test. wt, wild type.