The yersiniabactin transport system is critical for the pathogenesis of bubonic and pneumonic plague

Abstract

Iron acquisition from the host is an important step in the pathogenic process. While Yersinia pestis has multiple iron transporters, the yersiniabactin (Ybt) siderophore-dependent system plays a major role in iron acquisition in vitro and in vivo. In this study, we determined that the Ybt system is required for the use of iron bound by transferrin and lactoferrin and examined the importance of the Ybt system for virulence in mouse models of bubonic and pneumonic plague. Y. pestis mutants unable to either transport Ybt or synthesize the siderophore were both essentially avirulent via subcutaneous injection (bubonic plague model). Surprisingly, via intranasal instillation (pneumonic plague model), we saw a difference in the virulence of Ybt biosynthetic and transport mutants. Ybt biosynthetic mutants displayed an approximately 24-fold-higher 50% lethal dose (LD(50)) than transport mutants. In contrast, under iron-restricted conditions in vitro, a Ybt transport mutant had a more severe growth defect than the Ybt biosynthetic mutant. Finally, a Delta pgm mutant had a greater loss of virulence than the Ybt biosynthetic mutant, indicating that the 102-kb pgm locus encodes a virulence factor, in addition to Ybt, that plays a role in the pathogenesis of pneumonic plague.

FIG. 1.

Use of iron from transferrin (Tf) and lactoferrin (Lf). Growth responses of Ybt⁺ KIM6+ (A and C), Ybt⁻ KIM6-2046.1 (irp2::kan2046.1) (B), and KIM6-2046.3 (Δirp2-2046.3) (D) on PMH-EDDA plates to partially saturated Tf (1, 2, 5, and 6 in panels A and B), partially saturated bovine Lf (1, 2, 5, and 6 in panels C and D), or inorganic iron (3, 4, 7, and 8) are shown. The solutions were added to wells (A and B) or on filter discs (C and D) on seeded plates (1, 3, 5, and 7) or spotted onto a dialysis membrane overlaying the bacterial cells (2, 4, 6, and 8). After incubation with Tf or Lf at 37°C, plates were overlaid with TBA containing esculin and ferric citrate to visualize bacterial growth. The images are from one of two or more independent experiments that yielded similar results.

FIG. 2.

Removal of iron from iron-saturated human transferrin by Y. pestis. Tf was incubated overnight with iron-starved cultures of Y. pestis Ybt⁺ KIM6+ (lane 2) or Ybt⁻ KIM6-2046.1 (lane 3). Tf was in a dialysis baggy (12,000- to 14,000-Da molecular mass cutoff), separating it from the cultures. After incubation, the various iron-saturated Tf forms were separated on 6% polyacrylamide-urea gels. Incubation of Tf with PMH growth medium (lane 1) was used as a negative control. Partially saturated Tf (lane 4) and a mixture of various forms of Tf (lane 5) were used to show mobility shifts due to iron saturation of Tf. The image is from one of two independent experiments, which yielded similar results.

FIG. 3.

Time-to-death analysis from i.n. instillation studies. Except for the Δpgm mutant, infectious doses used were close to the calculated LD₅₀ for that strain. The average doses (in parentheses) were calculated from two (Ybt⁺ and psn), three (irp2), and four (Δpgm) independent experiments. All studies were carried out to 14 days, with no further deaths after day 10. Data are averages from all LD₅₀ studies, shown as percent survival on the indicated days postinfection.

FIG. 4.

Iron-deficient growth of Y. pestis strains. All strains were grown in deferrated PMH2 at 37°C. Where indicated, purified Ybt was added to KIM6+ (Ybt⁺) and KIM6-2180 (Δirp2-2046.3 Δpsn2045.1) at a concentration similar to that produced by a Ybt⁺ strain. The growth curves shown are from one of two independent experiments, which yielded similar results.