The role of relA and spoT in Yersinia pestis KIM5 pathogenicity

Abstract

The ppGpp molecule is part of a highly conserved regulatory system for mediating the growth response to various environmental conditions. This mechanism may represent a common strategy whereby pathogens such as Yersinia pestis, the causative agent of plague, regulate the virulence gene programs required for invasion, survival and persistence within host cells to match the capacity for growth. The products of the relA and spoT genes carry out ppGpp synthesis. To investigate the role of ppGpp on growth, protein synthesis, gene expression and virulence, we constructed a Delta relA Delta spoT Y. pestis mutant. The mutant was no longer able to synthesize ppGpp in response to amino acid or carbon starvation, as expected. We also found that it exhibited several novel phenotypes, including a reduced growth rate and autoaggregation at 26 degrees C. In addition, there was a reduction in the level of secretion of key virulence proteins and the mutant was > 1,000-fold less virulent than its wild-type parent strain. Mice vaccinated subcutaneously (s.c.) with 2.5x10(4) CFU of the Delta relA Delta spoT mutant developed high anti-Y. pestis serum IgG titers, were completely protected against s.c. challenge with 1.5x10(5) CFU of virulent Y. pestis and partially protected (60% survival) against pulmonary challenge with 2.0x10(4) CFU of virulent Y. pestis. Our results indicate that ppGpp represents an important virulence determinant in Y. pestis and the Delta relA Delta spoT mutant strain is a promising vaccine candidate to provide protection against plague.

Figure 1. Schematic chromosome structure of Y. pestis KIM6⁺, χ10003 (ΔrelA233), χ10004 (ΔrelA233ΔspoT85) and χ10019 (ΔrelA233 ΔspoT85 ΔlacZ::TT araC P_BAD spoT).

Figure 2. Analysis of (p)ppGpp synthesis in Y. pestis KIM6⁺ and ΔrelA ΔspoT mutants during amino acid and carbon starvation by TLC.

Total intracellular nucleotides were extracted from Y. pestis cultures uniformly labeled with [³²P] H₃PO₄. Cells were grown in modified PMH2 medium lacking L-phenylalanine for amino acid starvation (A) and in modified PMH2 medium without glucose for carbon starvation (B).

Figure 3. Growth of Y. pestis strains in HIB medium at different temperatures (A) Growth curve at 26°C; (B) Growth curve at 37°C. •, Y. pestis KIM5⁺; ▪, χ10003(pCD1Ap) (ΔrelA233) ▴, χ10004(pCD1Ap) (ΔrelA233ΔspoT85); ▾, χ10019(pCD1Ap) (ΔrelA233 ΔspoT85 ΔlacZ::TT araC P_BAD spoT) without arabinose; ♦, χ10019(pCD1Ap) (ΔrelA233 ΔspoT85 ΔlacZ::TT araC P_BAD spoT) with 0.05% arabinose.

Figure 4. Analysis of virulence factor expression and secretion in Y. pestis KIM5⁺ and mutants.

(A) Evaluation of virulence factor transcription by semi-quantitative RT-PCR. (B) Measurement of secreted virulence factors in culture supernatants by western blotting. Secreted proteins were collected from the culture medium following the removal of bacterial cells. Proteins were separated by SDS-PAGE and detected by western blotting. For each sample, the same amount of total protein was loaded.

Figure 5. Survival of Swiss Webster mice (3 mice per strain) infected s.c. with Y. pestis KIM5⁺ (black circles), χ10003(pCD1Ap) (black squares), χ10004(pCD1Ap) (black triangles) and χ10019(pCD1Ap) cultured with 0.05% arabinose in vitro (black diamonds).

The experiment was performed twice with similar results.

Figure 6. Kinetics of infection with Y. pestis KIM5⁺ (black) or χ10004(pCD1Ap) (white) in mouse tissues.

Groups of nine mice were inoculated s.c., and at various times CFU per organ in the blood (A), lungs (B), spleens (C) and livers (D) were determined for 3 mice per group. Error bars represent standard deviation.

Figure 7. Antibody response in sera of mice inoculated with Y. pestis KIM5⁺ or χ10004(pCD1Ap).

A Y. pestis whole cell lysate was used as the coating antigen. (A) Serum IgG responses. (B) Serum IgG1 and IgG2a responses. *, the P value was less than 0.01; **, the P value was less than 0.05.

Figure 8. Mouse survival after Y. pestis KIM5⁺ Challenge.

(A) Swiss Webster mice vaccinated s.c. with 2.5×10⁴ CFU of χ10004(pCD1Ap) and a were challenged with 1.5×10⁵ CFU of Y. pestis KIM5⁺ via the s.c. route. (B) Swiss Webster mice vaccinated s.c. with 2.5×10⁴ CFU of χ10004(pCD1Ap) were challenged via the i.n. route with 2×10⁴ CFU of Y. pestis KIM5⁺. Immunization provided significant protection against both challenge routes (P<0.001). For each experiment, there were 10 mice in the vaccinated group and 4 mice in the control group.

Figure 9. IL-10 production in sera of mice inoculated with Y. pestis KIM5⁺ or χ10004(pCD1Ap).

*, the P value was less than 0.01; **, the P value was less than 0.05.