Figure 1.
Incidence of plague in mice infected with Y. pestis lacking the zinc transporter Znu and/or the putative zinc murein DD-endopeptidase YebA (YPO2062).
(A), mice were injected intradermally with ∼10 Y. pestis WT (white circles) and ΔznuABC ypo2062 (black squares) or ΔznuC (black diamonds). (B), mice were inoculated with as in (A) with WT (white circles) or Δypo2062 (grey circles) mutants. Only the loss of YPO2062 significantly decreased the virulence of Y. pestis (p<0.05, relative to WT). Groups of 8 mice were used. Data obtained with the ΔznuABC ypo2062 mutant are representative of two experiments. Data obtained with the ΔznuC and Δypo2062 mutants come from one experiment each.
Table 1.
List of mutants selected using the per-pool screening method.
Figure 2.
Carbohydrate uptake and metabolism by Y. pestis in its mammalian host.
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 in vivo [7]. 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 in vivo 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.
Figure 3.
Incidence of plague in immunocompetent rats (A), immunocompetent mice (B), and neutropenic mice (C) injected intradermally with ∼20 WT Y. pestis (white circles), Δypmt1.66c (black circles) or complemented mutant (grey circles).
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 ypmt1.66c gene on a high copy number plasmid (pCR2) and the wild-type (B) were significantly different (p<0.05). The virulence of Y. pestis lacking ympt1.66c 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.
Figure 4.
The change over time in colonization and bacterial load in rats inoculated intradermally with ∼20 WT Y. pestis (white circles) or Δypmt1.66c (black circles).
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.
Figure 5.
YPMT1.66c is required for intracellular survival in macrophages (A) and for optimal growth in serum (B).
Shown are the data obtained with the wild-type (white squares and bars), the Δypmt1.66c (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).
Figure 6.
Stress, metabolic and uncharacterized genes are needed for resistance and/or growth in serum.
The ability of selected mutants to grow in fresh serum was evaluated after 6 and 21ΔrseC, Δypo0337, ΔgpmA, ΔibpA, Δypo3369, Δypo0988, Δamn, Δypo0617-0618, Δypo2586-2587 and Δypo0426 mutants differed significantly from that of the wild-type strain (p<0.05 in a two-way analysis of variance).