, 2008). HIF inhibitor In an attempt to identify the target proteins affected by virB, we compared
protein differences between a virB mutant and its parental strain using comparative proteomic analysis (Wang et al., 2009). Interestingly, several intracellular survival-related proteins, including VjbR, DnaK, HtrA, Omp25 and GntR, were downregulated in the virB mutant. Of these proteins affected by virB, products of the two major outer membrane proteins (OMPs), Omp25 and Omp31, were expressed at decreased levels, implying that T4SS might affect the membrane properties of Brucella. OMPs are essential for maintaining the integrity and selective permeability of membranes (Moriyon & Lopez-Goni, 1998). In addition, OMPs are often regulated by environmental signals and play important roles in bacterial pathogenesis by enhancing the adaptability to various environments (Lin et al., see more 2002; Caro-Hernandez et al., 2007). Virulence regulation systems, exemplified by VjbR and BvrR/BvrS, regulate the expression of membrane proteins. The mutants showed an altered
expression of OMPs. Because of the limited separation resolution of two-dimensional polyacrylamide gel electrophoresis (2-DE), only a small part of the proteins could be isolated and identified. Therefore, it is possible that far more OMPs are differentially expressed in the virB mutant and that OM-related phenotypes are altered. To further test the effect of T4SS on the OM, in the present study, OMPs of a wild-type and a virB Protein kinase N1 mutant strain were isolated and compared. The membrane integrity was tested by comparing the sensitivity of these proteins to polymyxin B and several stresses. Notably, a large number of OMPs were differentially expressed. More protein products of Omp25 and Omp31 were shown to be altered, revealing a complicated post-translational modification of the two proteins. In vitro sensitivity assays showed that the resistance of the virB mutant to different stress
environments was reduced. These data indicated that a drastic modification in the OM of the virB mutant occurred and that T4SS plays important roles in membrane integrity. A virB inactivation mutant BMΔvirB (BM with a promoter of the virB operon deleted) and complementary strains BM-IVGT (BMΔvirB containing complementary plasmid pBBR1-IVGT) were constructed previously (Wang et al., 2009). Brucella was cultured in tryptic soy broth (TSB) or tryptic soy agar (TSA). When necessary, antibiotics were added to a final concentration of 100 μg mL−1 ampicillin and 25 μg mL−1 gentamicin. The Brucella OM fractions were isolated as described previously (Ying et al., 2005). 2-DE and matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) MS were performed essentially as described previously (Wang et al., 2009). Total RNA was isolated with Trizol agent (Invitrogen, Carlsbad, CA) as recommended by the manufacturer.