FIG. 9.

LcrV-specific antibody does not neutralize Yop-targeting activity of Y. pestis preinduced for expression of LcrV and Yops. Y. pestis KIM8-3002 was grown overnight at 26°C, diluted into warm RPMI, centrifuged in six-well dishes, and incubated for 2 h at 37°C with 5% CO₂ to allow surface expression of LcrV. After 1.5 h of the incubation, the bacteria were resuspended, and α-YopM or α-HTV was added to some wells. The yersiniae were used to infect HeLa cells at an MOI of 2, and some wells were photographed at hourly intervals, up to 4 h, through phase-contrast optics and a green filter. (A) Cultures after 2 h of infection. (B) After 4 h of infection, replicate cultures were treated (+) or not (−) with trypsin, harvested, and fractionated as described in Materials and Methods, and TCA-precipitated proteins from cell-free medium (Med) and HeLa cell soluble (Sol) fractions were analyzed by immunoblotting with a mixture of α-HTV and α-YopE antibodies as a probe. The rightmost two lanes contain 0.2 and 2.0 μg of α-HTV as a reference for the size of the predominant band due to antibody. The proteins were visualized by probing with horseradish peroxidase-coupled secondary antibody followed by development with ECL reagent and exposure to film.