7B and 7C, lane 1). The data indicated that the NTUH-S1 strain expressed both the Lea and Leb antigens. In addition, the amounts of O-antigen (~34 kDa) in the imp/ostA or msbA single mutant were reduced, and it was especially reduced in the imp/ostA and msbA double mutant. The growth

curves of the wild-type and mutant strains were also examined, and the growth rates of these mutants did not differ from that of the wild-type strain (data not shown). This result demonstrated that both imp/ostA and msbA were involved in the production of LPS. Figure 7 Silver-stained of proteinase-K Dinaciclib clinical trial digested whole cell lysate from H. pylori wild-type and isogenic mutants. (A) Lanes 1–7 were all loaded with 2.5 × 108 proteinase K-digested bacteria (~130 μg total protein). Lane 1, 26695; lane 2, wild-type; lane 3, imp/ostA single mutant

strain; lane 4, imp/ostA complementation strain; lane 5, msbA single mutant strain; lane 6, msbA complementation strain; lane 7,imp/ostA and msbA double mutant strain. Molecular weights of the prestained markers are indicated. (B-C) Immunoblots of LPS from H. pylori with anti-Lea or anti-Leb monoclonal find more antibodies. (B) anti-Lea (1:3000) as the primary antibody and anti-mouse IgG (1:5000) as the secondary antibody, or (C) anti-Leb (1:3000) as the primary antibody and anti-mouse IgG (1:5000) as the secondary antibody. Outer membrane permeability to ethidium Selleck Talazoparib bromide To investigate whether the permeability of the outer membrane was altered in the mutant strains, we measured the fluorescence intensity at a 40-min time point after addition of ethidium bromide and CCCP (Fig. 8A). The fluorescence intensity of the imp/ostA deletion mutant O-methylated flavonoid (1142.73 ± 12.38 relative fluorescence units [RFUs]) was higher than that of the wild-type (891.29 ± 20.62 RFUs, P = 0.0001). The fluorescence intensity of the msbA deletion mutant was also significantly higher than the wild-type (P = 0.00164). These results might due to the increase of outer membrane permeability when imp/ostA or msbA was mutated. Furthermore, the fluorescence intensity of the imp/ostA and msbA double mutant was also significantly

higher than that of wild-type (P = 5.83 × 10-5). Therefore, the increased sensitivity to hydrophobic compounds conferred by imp/ostA and msbA mutations can be explained by the enhanced membrane permeability for the toxic substances moving in. Figure 8 Permeability and efflux of ethidium bromide. (A) Determination of the outer membrane permeability in H. pylori wild-type and isogenic mutants. Each measurement was repeated three times. *, P < 0.05 vs. wild-type, and **, P < 0.001 vs. wild-type. (B) Ethidium bromide accumulation assay. Cells were preloaded with 10 μg/ml ethidium bromide. At the 12-min time point, 10 μM of CCCP was added to the cells suspensions to assess energy-dependent efflux. CCCP was not added to the cells serving as controls (dotted lines).