Table 1.
Strains and Plasmids.
Table 2.
Primers.
Figure 1.
Y. pestis wild type and ΔtatA mutant strains were grown in HI broth at 28°C (A and B), 37°C with CaCl2 (C and D), or 37°C with MOX (E and F). Growth was monitored by both optical density (A, C, and E) and CFU counts (B, D, and F). OD measurements are based upon quadruplicate samples and bars represent standard deviation. CFU measurements are based upon triplicate samples and bars represent standard deviation. These data represent at least two separate experiments.
Figure 2.
Morphology of Y. pestis strains.
Y. pestis was grown at 37°C in presence CaCl2 and examined by microscopy. The samples, wild-type (A) and ΔtatA (B), were fixed, stained with Wayson stain, and examined by microscopy (100×). Additionally, samples, wild-type (C; micron bar = 0.5 µm) and ΔtatA (D; micron bar = 0.5 µm), were examined by TEM.
Figure 3.
Adherence of Y. pestis biofilm.
The Y. pestis A) wild-type CO92 and B) ΔtatA mutant cultures were grown in HI broth at 26°C. Arrows indicate biofilm formation. C) To measure biofilm adherence, the biofilm of wild-type CO92, ΔtatA mutant, and complemented ΔtatA mutant was stained with crystal violet and assayed by absorbance as described in the Materials and Methods. The standard deviation was derived from quadruplicate samples. These data represent three separate experiments.
Figure 4.
Analysis of Y. pestis proteins.
A) Strains (wild-type, lanes 1 and 4; ΔtatA, lanes 2 and 5, and C12, lanes 3 and 6) were grown in HI broth at 28°C or 37°C containing CaCl2, as indicated. Proteins were extracted and ran on a SDS-PAGE gel at equal concentrations. The arrow indicates a protein band that is synthesized at 37°C for the CO92 and to a lesser extent the ΔtatA mutant but not in C12, a F1-antigen mutant strain. Molecular masses are indicated on the left in KDa. Strains were grown in HI broth at 37°C containing CaCl2 and proteins extracted for Western analysis. Equal concentrations of proteins from Y. pestis strains were blotted with a monoclonal antibody to either the F1 antigen (B) or GroEL (C). Lanes: 1, wild-type; 2, ΔtatA; 3, C12; and 4, recombinant protein (F1 antigen or GroEL, respectively). Molecular masses are indicated on the left in KDa. D) Strains were grown in HI broth at 37°C in the presence of MOX to create low calcium conditions. Equal concentrations of proteins from Y. pestis strains were blotted with an antibody to LcrV Lanes: 1, CO92 wild-type; 2, ΔtatA; 3, Y. pestis pLcr-; and 4, rLcrV protein. Molecular masses are indicated on the left in KDa.
Figure 5.
Localizing the F1 capsule by microscopy.
Y. pestis strains were grown at 37°C in HI broth containing CaCl2 and exposed to an antibody against the F1 capsule. IFM samples: A) wild-type, B) ΔtatA mutant, and C) complemented ΔtatA mutant. The images (100×) were captured for all samples at identical camera settings to maintain relative fluorescence. IEM samples: D) wild-type (micron bar = 0.1 µm), E) ΔtatA mutant (micron bar = 0.1 µm), and F) C12, F1 negative (micron bar = 0.5 µm).
Figure 6.
Groups of Swiss Webster mice were challenged and survival monitored following various plague models of infection (sub-Q injections A–C; small-particle aerosol D and E; and intranasal F and G) with various strains of Y. pestis (wild-type A, D, and F; ΔtatA mutant B, E, and G; and the complemented ΔtatA mutant C). The calculated LD50 values are included in Table 3 3.
Table 3.
Calculated LD50 for the CO92 wild-type and ΔtatA mutant Y. pestis strains.
Table 4.
Survival and TTD comparisons between mice challenged intranasally with the ΔtatA mutant and wild-type Y. pestis strains.
Figure 7.
Pathology of mice challenged intranasally with Y. pestis.
A and B) Skull sections (4×) of a mouse challenged with the ΔtatA mutant (2,800 CFU). The mouse was moribund and euthanized on day 8 postchallenge. Note inflammation (indicated by *) that extends into the cranial vault and meninges. Panel A shows HE staining, and Panel B shows IHC staining with antibody to the F1 capsule. C) HE stained section from a mouse (2×) challenged with wild-type Y. pestis (5,500 CFU) that succumbed to infection on day 3 post challenge. In contrast to panel A, the inflammation for mice challenged with the wild-type strain, is contained in the inner ear area and did not extend into the brain (arrow). D and E) Lung sections of mice challenged intranasally with Y. pestis. Overall, lung lesion development and character are the same between strains with necrosuppurative inflammation surrounding large colonies of bacteria. Panel D is of a lung section stained with HE (20×) from a mouse challenged with the ΔtatA mutant. Panel E is of a lung section stained with HE (20×) challenged with wild-type Y. pestis. IE = inner ear; CRB = cerebrum.
Figure 8.
Pathology of mice challenged with Y. pestis by small particle aerosol.
A and B) HE stained skull sections of aerosol challenged mice. A) Mouse (2×) challenged with wild-type CO92 (6.9×105 CFU) that was moribund and euthanized on day 3 postchallenge. B) Mouse (4×) challenged with ΔtatA mutant (3.3×106 CFU) that was moribund and euthanized on day 7 postchallenge. No inner ear (IE) or meningeal involvement was detected for mice aerosol challenged with either strain of Y. pestis. HE stained lung sections (10×) of mice aerosol challenged with CO92 (C) or ΔtatA mutant (D). There was no difference in lesion character or severity in mice challenged with either strain.
Figure 9.
Dissemination studies of mice challenged intranasally with Y. pestis.
Mice were challenged with CO92 (113,000 CFU) or ΔtatA mutant (76,000 CFU). At set time points, mice were euthanized, and the lungs and spleens were harvested. The A) lungs and B) spleens were homogenized and plated to determine bacterial recovery. For each time point, five mice were assayed, except for day 3 for wild-type challenged mice. Only four mice were tested as the remaining challenged mice had succumbed to infection. The lines (solid = CO92 and hashed = ΔtatA) are connecting at the geometrical means at the data points of CFU recovery from the respective organs are represent the overall trend during the course of infection.
Table 5.
Y. pestis proteins containing putative Tat motifs.