Fig. 2.

Colonization, antiphagocytosis activity and MARTX expression of the Δlrp mutant. a The bacteria (2 × 10⁶ cfu) were injected into an air sac on the back of a normal or neutropenic mouse. The mice were sacrificed 6 h after infection, and the bacteria collected from the air sac were enumerated by viable counts. n = 3. YJ016: wild-type strain; YH01: Δlrp mutant. The results of no significant difference analyzed by one-way ANOVA along with Tukey’s test are labeled with the same letters, and those of significant differences (P < 0.05) are labeled with different letters. b A mixture of equal numbers of mutant YH01 and wild-type strain CP212 (2 × 10⁶ cfu totally) were injected into the air sac on the back of a normal mouse. The mice were sacrificed 6 h after infection, and the viable bacteria collected from the air sac were enumerated by viable counts. n = 3. Strain CP212 was distinguished from mutant YH01 (white vs. blue colonies) on an X-gal-containing plate. c and d RAW 264.7 cells were cocultured with the bacteria at a moi of 10 for 30 min. At each time point, the intracellular bacteria number c was enumerated by viable counts after addition of gentamicin to kill the extracellular bacteria, and the survival rate of the internalized bacteria in d was calculated as described in ‘Phagocytosis assay’ of ‘Methods’. n = 3. YJ016: wild-type strain; HL128: ΔrtxA1 mutant; YH01: Δlrp mutant. The significance of difference was analyzed by t-test and two-way ANOVA with Bonferroni’s post test, respectively, for c and d. ***: P < 0.001 for the difference between YH01 and YJ016 or HL128. e and f Total cell lysates were prepared from the bacteria collected from a 4 h culture in LB broth e or RAW 264.7 cells cocultured with the bacteria at a moi of 10 for 1.5 h at 37 °C f. The proteins in the cell lysate were fractionated by electrophoresis on a 6% SDS-polyacrylamide gel and then subjected to Western blot analysis with anti-ERM antiserum. The upper panel in e is the result of Western blot analysis; the lower panel is the result of Coomassie blue stain of one of the duplicated gels to show that similar amount of proteins was loaded in each lane. β-actin was used as an internal control in f. YJ016: wild-type strain; HL128: ΔrtxA1 mutant; NY303: spontaneous attenuated mutant; YH01: Δlrp mutant; YH03: YH01(plrp)