, 2008). The major component in the outer monolayer of the BTK inhibitor concentration OM in gram-negative bacteria is lipopolysaccharide. Lipopolysaccharide is a complex glycolipid
composed of lipid A, core oligosaccharide, and the O-specific polysaccharide chain. We observed in this study that many genes required for the biosynthesis (lpxDAB, lpxC, lpxH, and msbB2), transportation (crcA) and modification (msbA) of lipid A were significantly upregulated. Among these genes, msbB2 and crcA are known to be induced by a lack of Mg2+ (Guo et al., 1998; Goldman et al., 2008). Therefore, it is likely that Mg2+ in the envelope may be limited due to BC treatment. Divalent cations such as Mg2+ are absolutely required for the activity of MdoB, which aids in generating a net negative charge in membrane-derived oligosaccharides
to maintain periplasmic osmolarity (Jackson & Kennedy, 1983). We found that the gene encoding the MdoB protein was induced, indicating that MdoB activity may be inhibited due to the lack of divalent cations, which may in turn disturb the periplasmic osmolarity. In accordance with this suggestion, some high-osmolarity-inducible OM genes were downregulated by the drug, including blc, bolA, yehZ and osmB. The induction of lpxC and BYL719 in vivo repression of blc, bolA and yehZ were also monitored in the QRT-PCR assay. Many studies have shown that cationic peptides have high affinities for divalent cation-binding sites in the lipopolysaccharide, and they therefore easily displace the cations, which are known to be essential for maintaining OM integrity (Hancock & Lehrer, 1998). Berberine alkaloids are amphipathic cations.
We therefore propose that BC may competitively displace divalent cations of the lipopolysaccharide, resulting in the limitation of Mg2+. The increased synthesis and transportation of lipid A may represent an adaptive response of Shigella to OM stress caused by BC. In Salmonella typhimurium and E. coli, the PhoQ–PhoP Unoprostone system confers resistance to cationic peptides by lowering the overall negative charge of lipopolysaccharide (Groisman et al., 1997). The expression of several PhoP-activated genes such as crcA (also known as pagP) and yhjw was increased, indicating that the PhoQ–PhoP system was weakly induced at concentrations well below the MIC of BC. Consistent with our results, a recent study has shown that a suprainhibitory concentration (10 × MIC) of berberine can not only increase the transcription of some genes required for the biosynthesis of lipopolysaccharide but also enhance the level of phoQ and Mg2+ transport protein encoded by mgtC (Zhang et al., 2009).