Figure 1.
Distinct transcriptional profile of Y. pestis in infected fleas.
(A) Principal Component Analysis (PCA) representation of replicate microarray gene expression profiles of Y. pestis KIM6+ from blocked fleas (blue symbols) and from in vitro flowcells, exponential phase planktonic cultures, and stationary phase planktonic cultures (red, green, and purple symbols, respectively). (B) Venn diagrams representing the number of Y. pestis genes upregulated or downregulated ≥2-fold in the flea relative to in vitro culture conditions.
Figure 2.
Y. pestis amino acid uptake and catabolism pathways upregulated in the flea.
Periplasmic and inner membrane uptake proteins for proline (PutP), histidine (HisJMPQ), glutamine (GlnHPQ), arginine (ArtIMPQ), spermidine (PotABCD), and hydroxyphenylacetate (HpaX) are indicated from left to right. Genes encoding catabolic enzymes leading to glutamate and TCA cycle intermediates are also shown. Symbols labeled in blue indicate genes upregulated ≥2-fold in the flea compared to all in vitro conditions (Table S1).
Figure 3.
Distinct Y. pestis gene expression profiles in flea and rat hosts.
(A) Hierarchichal clustering of normalized microarray data sets of Y. pestis gene expression in the rat bubo and the flea. The scale indicates relative transcript levels (blue = low; red = high) for all 4,638 Y. pestis genes on the microarray. (B) Percentages of Y. pestis genes that are differentially regulated (or not) in the flea and in the rat bubo.
Table 1.
Differential expression of Y. pestis pathogenesis-related genes in the flea.
Figure 4.
Phagocytosis-resistant phenotype of Y. pestis isolated from fleas correlates with expression level of the yit-yip insecticidal-like toxin genes.
(A) The percentage of extracellular Y. pestis KIM6+ 1 hour after addition to murine bone marrow macrophages are shown for bacteria from in vitro cultures (LB) or from infected fleas. The mean and SEM of five independent experiments done in duplicate are shown; P<0.0001. (B) Relative transcript levels of insecticidal-like toxin genes in Y. pestis KIM6+ wt grown in LB (black bars), ΔyitR mutant grown in LB (grey bars), ΔyitR mutant from fleas (white bars), and the complemented ΔyitR mutant from LB (hatched bars) and from fleas (green bars); nd = not done. The mean and SEM of three independent experiments done in triplicate are shown. Values corresponding to separate segments of the y-axis are significantly different (P<0.001); values for LB-grown wt bacteria (black bars) are also significantly different from values represented by the grey and white bars (P<0.05). (C) Differential resistance to phagocytosis by murine macrophages (% extracellular flea-derived bacteria minus % extracellular in vitro-grown bacteria) of Y. pestis KIM6+ wt (black bar, n = 3), ΔyitR mutant (white bar, n = 3), and complemented ΔyitR mutant (grey bar, n = 2). The mean and standard error of the n experiments done in duplicate are indicated; *P<0.01; ** P = 0.06.
Figure 5.
Mean and range of the cumulative number of blocked flea bites received by mice.
Circles and squares indicate individual mice challenged by fleas infected with wt or ΔyitR Y. pestis 195/P, respectively. Filled symbols indicate mice that developed terminal plague; open symbols indicate mice that did not develop disease.