New Insights into How <i>Yersinia pestis</i> Adapts to Its Mammalian Host during Bubonic Plague

<div><p>Bubonic plague (a fatal, flea-transmitted disease) remains an international public health concern. Although our understanding of the pathogenesis of bubonic plague has improved significantly over the last few decades, researchers have still not been able to define the complete set of <i>Y. pestis</i> genes needed for disease or to characterize the mechanisms that enable infection. Here, we generated a library of <i>Y. pestis</i> mutants, each lacking one or more of the genes previously identified as being up-regulated <i>in vivo</i>. We then screened the library for attenuated virulence in rodent models of bubonic plague. Importantly, we tested mutants both individually and using a novel, “per-pool” screening method that we have developed. Our data showed that in addition to genes involved in physiological adaption and resistance to the stress generated by the host, several previously uncharacterized genes are required for virulence. One of these genes (<i>ympt1.66c</i>, which encodes a putative helicase) has been acquired by horizontal gene transfer. Deletion of <i>ympt1.66c</i> reduced <i>Y. pestis</i>' ability to spread to the lymph nodes draining the dermal inoculation site – probably because loss of this gene decreased the bacteria's ability to survive inside macrophages. Our results suggest that (i) intracellular survival during the early stage of infection is important for plague and (ii) horizontal gene transfer was crucial in the acquisition of this ability.</p></div

Incidence of plague in immunocompetent rats (A), immunocompetent mice (B), and neutropenic mice (C) injected intradermally with ∼20 WT <i>Y. pestis</i> (white circles), Δ<i>ypmt1.66c</i> (black circles) or complemented mutant (grey circles).

<p>The mutant was significantly (p<0.05) less virulent than the WT strain in immunocompetent rats (A), immunocompetent mice (B and C) and neutropenic mice (C). The survival curves of mice infected with the complemented mutant harboring the <i>ypmt1.66c</i> gene on a high copy number plasmid (pCR2) and the wild-type (B) were significantly different (p<0.05). The virulence of <i>Y. pestis</i> lacking <i>ympt1.66c</i> was no greater in neutropenic mice than it was in immunocompetent mice (dashed lines) (p>0.05). Data were obtained from groups of 8 or 9 animals. P values were determined using the Gehan-Breslow-Wilcoxon test.</p

Stress, metabolic and uncharacterized genes are needed for resistance and/or growth in serum.

<p>The ability of selected mutants to grow in fresh serum was evaluated after 6 and 21Δ<i>rseC</i>, Δ<i>ypo0337</i>, Δ<i>gpmA</i>, Δ<i>ibpA</i>, Δ<i>ypo3369</i>, Δ<i>ypo0988</i>, Δ<i>amn</i>, Δ<i>ypo0617-0618</i>, Δ<i>ypo2586-2587</i> and Δ<i>ypo0426</i> mutants differed significantly from that of the wild-type strain (p<0.05 in a two-way analysis of variance).</p

Survival of <i>Y. pestis</i>-infected mice treated with GEN alone, CIN alone and a combination of CIN+GEN.

<p>Groups of 19–20 mice were i.d. infected with 100 CFU of <i>Y. pestis</i> and were given antibiotics i.v., either alone or in combination and once or twice daily (depending on the antimicrobial). The first dose of drugs was given 44 h (experiment A) or 56 h (experiment B) after the <i>Y. pestis</i> inoculation.</p>a<p>Time point: 0, day of initiation of treatment; 1, one day after initiation of treatment; 5, the end of treatment-course; 10, euthanasia of survivors.</p

The effect of antibiotic treatment on bacterial load in the draining lymph node.

<p>Treatment with GEN at 25 mg/kg q24h (filled circles), CIN at 30 mg/kg q12h (open circles) and CIN at 30 mg/kg q12h in combination with GEN at 25 mg/kj q24h (filled diamonds) was initiated in mice 44 h (A) and 56 h (B) after the i.d challenge (100 CFU of <i>Y. pestis</i>). Individual bacterial counts in the draining lymph node 5 days after the end of the course regimen are shown. Median values are indicated by bars. The dashed horizontal line represents the LOD (≤1.8 log₁₀CFU).</p

Activities of CIN and GEN in combination against <i>Y. pestis</i> CO92.

<p>CIN and GEN were respectively used at (A) 0.016 mg/L and 0.5 mg/L (0.5x MIC), (B) 0.032 mg/L and 1 mg/L (1x MIC) and (C) 0.064 mg/L and 2 mg/L (2x MIC). The dashed horizontal line indicates the LOD (≤1.6 log₁₀ CFU).</p

YPMT1.66c is required for intracellular survival in macrophages (A) and for optimal growth in serum (B).

<p>Shown are the data obtained with the wild-type (white squares and bars), the Δ<i>ypmt1.66c</i> (grey squares and bars) and the complemented mutant (black squares and bars) strains. Data are quoted as the mean (SD) from three independent experiments using macrophages (A) and 5 independent experiments using serum from 5 different healthy donors (B). The mutant's survival (A) and growth curves (B) differed significantly from those of the WT and the complemented strain (p<0.05 in a two-way analysis of variance). The complemented mutant's and the WT's survival (A) and growth curves (B) did not differ significantly (p>0.05 in a two-way analysis of variance).</p

Carbohydrate uptake and metabolism by <i>Y. pestis</i> in its mammalian host.

<p>Genes encoding transporters for fructose (FruAB), mannose (ManZYX), maltose (MalEMGKF and MalE₂FG), glucose (PtsG), gluconate (GntM) and fructuronate (GntP) and the carbohydrate metabolism genes shown in red or purple were all overexpressed <i>in vivo </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004029#ppat.1004029-Sebbane1" target="_blank">[7]</a>. In virulence tests, mutants lacking a gene highlighted in red were virulence-attenuated, whereas those lacking a gene highlighted in purple were not. Genes in grey were not upregulated <i>in vivo</i> and their role in virulence has yet to be elucidated; hence, the importance of the metabolic fluxes involving the corresponding enzymes in virulence is unknown (grey arrows). Red and purple arrows respectively indicate the metabolic fluxes considered to be important and non-essential for virulence. Yellow arrowheads represent the metabolic pathways that glucose and gluconate might follow.</p

List of mutants selected using the per-pool screening method.

†<p>, data were obtained from groups of 10 rats inoculated intradermally with a pool of 5 mutants (with 20 CFU of each mutant).</p

The change over time in colonization and bacterial load in rats inoculated intradermally with ∼20 WT <i>Y. pestis</i> (white circles) or Δ<i>ypmt</i>1.66c (black circles).

<p>N.D: not determined. Each circle represents one animal. The times to colonization were significantly longer in rats infected with the mutant than in rats infected with the WT strain (p<0.05). P values were determined using 2-way analysis of variance.</p