PMC

Enhancement of His₆-Pla-mediated plasmin formation by smooth and rough LPSs from Y. pseudotuberculosis, Y. pestis, and E. coli. Both His₆-Pla and the LPSs were tested at 200-pmol concentrations. LPS types are indicated on the figure. The assay was repeated three times, and means and standard deviations in a representative assay with triplicate samples are shown.

Plasminogen activation by purified His₆-Pla reactivated with LPS preparations from Y. pestis strains KM218, KM260, and 1146 cultivated at 25°C or at 37°C. The amount of the His₆-Pla protein and the LPSs was 200 pmol, and plasmin formation was measured as breakdown of the chromogenic substrate S-2251. The assay was repeated three times, and means and standard deviations in a representative assay with triplicate samples are shown.

Effect of the acylation level of LPS on reactivation of His₆-Pla. His₆-Pla was reactivated with hexa- and penta-acyl forms of Re and lipid A of E. coli. The assay was repeated three times, and means and standard deviations in a representative assay with triplicate samples are shown. Pla and the LPSs were tested at 200-pmol concentrations, and incubation was for 2 h. The LPS types were penta-acyl Re (bar a), hexa-acyl Re (bar b), penta-acyl lipid A (bar c), and hexa-acyl lipid A (bar d). Bar e shows activity of Pla with no added LPS.

Effect of substitution of the LPS-binding motif in Pla on plasminogen activation and Pla expression. The arginine residues R138 and R171 were replaced with glutamate individually or together in His₆-Pla. (A) The purified His₆-Pla proteins were reactivated with Y. pestis KM218 LPS. Plasmin formation by the His₆-Pla proteins is shown; the protein constructs are indicated on the figure. The pla constructs were cloned into the vector pSE380 and expressed in Y. pestis KIM D34 (B) or in E. coli XL1 (C), and plasmin formation by the recombinant bacteria is shown. The assays were repeated three times; means and standard deviations in a representative assay with triplicate samples are shown. (D) Western blotting of KIM D34 recombinant strains (7.5 × 10⁶ cells) with anti-His₆-Pla immunoglobulins as primary antibodies. The bacteria were the following: lane a, KIM D34(pSE380), lane b, KIM D34(pMRK1) expressing Pla; lane c, KIM D34 with Pla(R138E); lane d, KIM D34 with Pla(R171E); and lane e, KIM D34 with Pla(R138E/R171E).

Expression of the Pla protein, formation of β-Pla, and plasminogen activation by Y. pestis KIM D27 cells cultivated at 20°C or 37°C. Plasmin formation was measured as a breakdown of the chromogenic plasmin substrate. The assay was repeated three times, and means and standard deviations in a representative assay with triplicate samples are shown. ▪, plasmin formation by 8 × 10⁷ Y. pestis cells grown at 37°C; □, plasmin formation by 8 × 10⁷ Y. pestis cells grown at 20°C; ⋄, plasmin formation by Y. pestis cells from 20°C and adjusted to have the same amount of Pla protein as bacteria from growth at 37°C (i.e., 1.4 × 10⁸ cells). The inset shows Western blotting of 2 × 10⁷ Y. pestis cells with anti-Pla antiserum; the isoforms of the Pla molecule are indicated on the left. Plasmin formation by cells from 20°C remains low at both cell densities.