The Role of relA and spoT in Yersinia pestis KIM5+ Pathogenicity

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

<b>Bacterial strains and plasmids used in this study.</b>

a<p>In genotype descriptions, the subscripted number refers to a composite deletion and insertion of the indicated gene. P, promoter; TT, T4 ip III transcription terminator; <i>ori</i>, origin of replication; Kan^R, kanamycin resistance; Str/Spc^R, streptomycin/spectinomycin resistance; Amp^R, ampicillin resistance.</p

Gastric pH following histamine injection.

<p>Following a 6 h fast, mice were injected at time 0 with 10/kg histamine. The pH of the gastric contents was monitored for 4 h post histamine injection. Data shown are the mean and standard error of the mean of at least five mice per time point.</p

Survival of strains cultured under non-acid resistance inducing conditions.

<p>Wild-type enteric strains were grown in LB medium to late-log phase under aerobic conditions. <b>(A)</b> Cells were challenged in EG medium (pH 3.0) containing 0.1% casamino acids. Survival during EG medium challenge was assayed hourly for 4 h by plating onto LB agar. Data shown are the mean and SEM of three independent experiments. <b>(B)</b> Mice were either fasted for 6 h (fasted mouse model) or fasted and low gastric pH was induced by histamine injection (histamine mouse model) and then inoculated with 10⁹ CFU of each strain. Sixty min after inoculation, mice were euthanized and the entire small intestine removed and homogenized. Strain survival was assayed by plating onto LB agar containing kanamycin. Data are expressed as the percent of initial inoculum recovered (% survival). The geometric mean and 95% confidence interval of two independent experiments (8 mice total) is depicted.</p

Visualization of the bacterial inoculum in the gastrointestinal tract via light emission.

<p>Low gastric pH was induced in mice by histamine injection prior to inoculation with 1×10⁹ CFU of <i>S.</i> Typhi Ty2(pGEN-<i>luxCDABE</i>). At 30, 60 and 90 min post-inoculation, the gastrointestinal tract was removed and examined for the production of light by luciferase. A visible signal is equivalent to approximately 5×10⁵ CFU. Luminescence is reported as the number of photons detected per s per square cm. Images shown are representative results from groups of seven mice.</p

Effect of arginine decarboxylase on the gastric survival of <i>S.</i> Typhi.

<p>Pairs of attenuated <i>S.</i> Typhi strains differing only in their arginine decarboxylase locus were grown to stationary phase in EGA medium under aerobic conditions. Cells were combined in a 1:1 ratio in PBS containing 1 mM arginine. Low gastric pH was induced by histamine injection in mice fasted for 6 h. Mice were inoculated with 10⁹ CFU of each strain. Sixty min after inoculation, mice were euthanized and the entire small intestine removed and homogenized. Strain survival was assayed by plating onto LB agar containing kanamycin or streptomycin. Data shown are the competitive index of the two strains in each mouse with the geometric mean of two independent experiments (10 mice total) indicated as a solid line.</p

Correlation between in vitro and in vivo survival.

<p>The log₁₀ of the geometric mean number of CFU that survived in vitro challenge at pH 3.0 for 2 h was plotted against the log₁₀ of the geometric mean number of CFU recovered from the intestinal tissue in the <b>(A)</b> histamine mouse model or <b>(B)</b> fasted mouse model. Linear regression was performed on each data set and the r² value of the best-fit line is depicted for each model.</p

Characteristics of selected <i>Escherichia coli</i> isolates from chicken fecal samples used for <i>in vivo</i> experiments.

<p>Characteristics of selected <i>Escherichia coli</i> isolates from chicken fecal samples used for <i>in vivo</i> experiments.</p

Ability of fecal <i>Escherichia coli</i> isolates to urinary tract infection.

<p><i>E</i>. <i>coli</i> isolates MM242, MM243, MM244, MM248, and MM259, positive controls CFT073 and UTI89, and negative control MG1655 were assessed for their ability to colonize the (A) bladder and (B) kidney, and to invade in the (C) liver and (D) spleen of CBA/J mice. Mice were challenged with 10⁸ CFU via a urethral catheter and monitored for 2 days. Each experimental group contained at least 9 mice. Each dot represents an individual animal; the vertical dashed line separates chicken fecal <i>E</i>. <i>coli</i> isolates from control strains. An asterisk (*) represents significantly higher mean values determined by an ANOVA followed by Dunnett’s method for fecal <i>E</i>. <i>coli</i> isolates and positive control strains compared with the negative control.</p

Prevalence of virulence-associated <i>in vitro</i> phenotypes among chicken fecal <i>Escherichia coli</i> isolates.

<p>Prevalence of virulence-associated <i>in vitro</i> phenotypes among chicken fecal <i>Escherichia coli</i> isolates.</p