Temperature-dependence of yadBC phenotypes in Yersinia pestis

Abstract

YadB and YadC are putative trimeric autotransporters present only in the plague bacterium Yersinia pestis and its evolutionary predecessor, Yersinia pseudotuberculosis. Previously, yadBC was found to promote invasion of epithelioid cells by Y. pestis grown at 37 °C. In this study, we found that yadBC also promotes uptake of 37 °C-grown Y. pestis by mouse monocyte/macrophage cells. We tested whether yadBC might be required for lethality of the systemic stage of plague in which the bacteria would be pre-adapted to mammalian body temperature before colonizing internal organs and found no requirement for early colonization or growth over 3 days. We tested the hypothesis that YadB and YadC function on ambient temperature-grown Y. pestis in the flea vector or soon after infection of the dermis in bubonic plague. We found that yadBC did not promote uptake by monocyte/macrophage cells if the bacteria were grown at 28 °C, nor was there a role of yadBC in colonization of fleas by Y. pestis grown at 21 °C. However, the presence of yadBC did promote recoverability of the bacteria from infected skin for 28 °C-grown Y. pestis. Furthermore, the gene for the proinflammatory chemokine CXCL1 was upregulated in expression if the infecting Y. pestis lacked yadBC but not if yadBC was present. Also, yadBC was not required for recoverability if the bacteria were grown at 37 °C. These findings imply that thermally induced virulence properties dominate over effects of yadBC during plague but that yadBC has a unique function early after transmission of Y. pestis to skin.

Fig. 1.

yadBC does not affect infection dynamics in systemic plague. Y. pestis strains CO92.S38 (ΔyadBC; otherwise conditionally virulent; squares) and CO92.S42 (yadBC⁺ by restoration of the native yadBC operon; otherwise conditionally virulent; circles) were grown either at 28 °C (solid symbols) or at 28 °C and shifted to 37 °C for 3 h (open symbols). Groups of mice were infected intravenously in the retro-orbital plexus with 400 c.f.u. and were analysed for bacterial burdens in liver and spleen on the indicated days post-infection. The data were pooled and averaged from two experiments and represent between six and eight mice per datum point.

Fig. 2.

YadB and YadC do not affect early survival of Y. pestis in systemic plague. Pgm⁻ Lcr⁺ Y. pestis strains CO92.S38 (ΔyadBC; open bars) and CO92.S42 (yadBC⁺ by restoration of the native yadBC operon; solid bars) were grown as indicated to induce expression of thermally regulated proteins 3 h prior to infecting mice by the intravenous route. Groups of mice were infected intravenously with doses ranging from 5×10³ to 10⁴ bacteria. After 3 h, the recovery of viable bacteria was determined in livers and spleens and is presented as the fraction of input c.f.u. that was recovered. The data for the 28 °C/37 °C_3h growth protocol are pooled from four replicate experiments, and each datum point represents the mean±sd for 14 mice. Two experiments were done for yersiniae grown at 37 °C, and each datum point represents the mean±sd of pooled data from seven mice.

Fig. 3.

yadBC confers a phenotype in skin. Pgm⁻ Lcr⁺ Y. pestis strains differing in the presence of the yadBC operon and activity of the Pla protease (CO92.S38 and CO92.S42; CO92.S39 and CO92.S43) were grown at 28 °C, and doses of approximately 5×10³ bacteria were injected intradermally into C57BL/6 mice. For comparison, the Pla⁺ pair of strains also was given 3 h at 37 °C prior to infection (28 °C/37 °C_3h). After 3 h, the infected skin was removed, homogenized and plated for viable bacterial numbers. Two additional ΔyadBC/yadBC⁺ pairs were grown at 28 °C and tested for recovery to assess the importance of the Lcr plasmid and pgm locus on the yadBC phenotype. These were the Pgm⁺ Lcr⁻ Pla⁺ strains CO92.S15 and CO92.S8 and the Pgm⁻ Lcr⁻ Pla⁻ strains CO92.S39 and CO92.S43. The recovery of viable numbers was normalized to the input dose for each strain and is presented as mean±sd. Open bars, ΔyadBC Y. pestis; closed bars, reconstituted strain (ΔyadBC/yadBC⁺). The data were pooled from multiple experiments: 4–6 experiments (14–23 mice per datum point) for the Pgm⁻ strains grown at 28 °C, two experiments (eight mice per datum point) for the Pgm⁺ strains, and four experiments (14–16 mice per datum point) for the Pgm⁻ strains grown at 28 °C/37 °C_3h. Statistically significant differences are indicated; n.s., not statistically significantly different.

Fig. 4.

yadBC may dampen recruitment of PMNs. (a) Groups of mice were infected intradermally for 3 h with approximately 2×10³ Pgm⁻ Lcr⁺ ΔyadBC Y. pestis CO92.S38 and yadBC-reconstituted CO92.S42 strain grown at 28 °C (open and closed bars, respectively). After 3 h, the infected skin was removed, dissociated and plated for c.f.u. The recovery of viable numbers was normalized to the input dose for each strain and is presented as the mean±sd. The mock and α-Ly6G mice were given, respectively, 200 µg of rat IgG (mock ablation) or anti-Ly6G (to ablate PMNs) intraperitoneally 24 h prior to infection and immediately prior to infection. The bars represent 16 anti-Ly6G-treated and nine mock-treated mice infected with ΔyadBC Y. pestis CO92.S38 and 14 infected anti-Ly6G-treated and 10 mock-treated mice infected with Y. pestis CO92.S42. (b) Groups of three mice were infected intradermally for 3 h with approximately 2×10³ Pgm⁻ Lcr⁺ ΔyadBC Y. pestis CO92.S38 or yadBC⁺-reconstituted CO92.S42 bacteria grown at 28 °C (open and closed bars, respectively), and message abundance for the PMN marker Ly6G and the chemokines CXCL1 and CXCL2 were determined by RT-PCR in the dissociated skin and normalized to the message level of UbC (ubiquitin C). Skin from two uninfected mice (striped bars) was similarly analysed. The data were pooled from two experiments (six mice per datum point, except for uninfected mice, for which data from one mouse from each of the two experiments were pooled). Data are given as mean±sd. Statistically significant differences are indicated; n.s., not statistically significantly different.