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 ΔrelA ΔspoT 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°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 2.5×104 CFU of the ΔrelA ΔspoT mutant developed high anti-Y. pestis serum IgG titers, were completely protected against s.c. challenge with 1.5×105 CFU of virulent Y. pestis and partially protected (60% survival) against pulmonary challenge with 2.0×104 CFU of virulent Y. pestis. Our results indicate that ppGpp represents an important virulence determinant in Y. pestis and the ΔrelA ΔspoT mutant strain is a promising vaccine candidate to provide protection against plague.

ppGpp Conjures Bacterial Virulence

“…Additionally, the lethal dose of relA spoT mutant bacteria required to kill 50% of mice (LD 50 ) is ϳ100,000-fold greater than the LD 50 of WT and relA mutant Y. pestis bacteria. Moreover, the experimental induction of spoT fully complements the virulence defect of ppGpp 0 bacteria (190). relA spoT mutants exhibit WT colonization patterns in the blood, spleen, and liver; in contrast, the stringent response pathway may play a modest role in dissemination into the lungs, as judged by enumerating CFU in mouse tissues 3 days after infection.…”

“…pestis requires RelA to respond to amino acid starvation and SpoT to respond to carbon starvation (190). A source of ppGpp is critical for optimal Y. pestis growth in heart infusion broth, as relA spoT double mutants, but not relA single mutants, exhibit an increased lag phase and do not achieve WT cell densities at either 26°C (typical of fleas) or 37°C.…”

“…Doublemutant bacteria also demonstrate increased autoaggregation at 26°C but not at 37°C. Excess autoaggregation is due to the absence of spoT, as the expression of spoT in trans is sufficient to reduce the enhanced autoaggregation phenotype in a dosedependent manner (190).…”

“…pestis also requires a source of ppGpp in a mouse model of bubonic infection. After subcutaneous infection with either WT or relA mutant bacteria, mice die synchronously, with 100% mortality by 8 days postinfection (190). SpoT-dependent ppGpp levels are critical for lethality, as mice infected with relA spoT mutant Y. pestis cells exhibited a decreased mortality rate, with 80% of mice surviving at 6 days.…”

“…While expression studies by reverse transcription (RT)-PCR analysis of Yops (Yersinia outer proteins) and other T3SS effectors reveal WT patterns of transcription in relA mutant and relA spoT double-mutant bacteria, secreted protein levels are reduced in relA spoT mutants but not relA bacteria (190). Therefore, SpoT is sufficient to elicit a posttranscriptional mechanism that induces the expression of Y. pestis virulence factors directly or indirectly.…”