The Antibacterial Mechanism Of Houttuyfonate Homologues Against G~+ Bacteria

Houttuyninum naturally existing in traditional Chinese herb medicine Houttuynia Cordata Thunb and its derivate, houttuyfonate, had long been reported to effectively inhibit the growth of bacteria, such as S. aureus, M. catarrhalis, B. influenzae, S. pneumoniae and so on[9,10]. According to the molecular structure of houttuyninum (HOU-C10), a series of houttuyfonate homologues (HOU-Cn, and HOU-C18:n) were synthesized. The antibacterial test indicated that gram-positive (G+-) bacteria were more sensitive to HOU-Cn, than gram-negative (G'-) bacteria, and the sensitivity increased with the elongation of hydrocarbon chain of HOU-Cn. The fluorescence experiments indicated that HOU-Cn directly bound to membrane proteins of G+-bacteria by hydrophobic interaction, and exerted a stronger antibacterial activity. But it is not clear how to inhibit the growth of bacteria.In this study, Bacillus subtilis was used as the model bacterium, the effects of HOU-Cn on the bacterial surface by SEM, the permeability and fluidity of cell membrane, the conformation of membrane protein, the distribution of HOU-Cn, composition of fatty acids and the activity of FASâ…¡were investigated to clarify the antibacterial mechanism of HOU-Cn.1 The effect of HOU-Cn on the morphology of Bacillus subtilisThe Scanning electron microscope (SEM) results showed that during the period of bacterial growing, HOU-Cn could inhibit the synthesis of glycocalyx, and further made the glycocalyx on the surface of B.subtilis disappeared.Glycocalyx can only be found out of the cell wall of bacteria.Capsule can be formed by glycocalyx, which could prevent bacterial from the chemical injury.The disappearance of glycocalyx lead to the reduce of supply of nutrients.And the adaptation of bacteria to the environment also weakened accordingly.2 The effect of HOU-Cn on the cell membrane permeability, electrical conductivity and membrane protein of Bacillus subtilisThe results of fluorescence experiments indicated that HOU-Cn could influence the structure of membrane protein by interacting the proteins on the cell membrane or cell wall of the bacteria.The bacterial treated with HOU-Cn indicated the electric conductivity of bacteria and the fluidity of membrane lipidin creased gradually with the elongation of aliphatic chain. HOU-Cn could change Bacillus subtilis membrane protein function by changing the conformation of membrane protein. The drophobic binding between HOU-Cn and membrane made the effctive concentration of HOU-Cn on membrane increased greatly. With the elongation of carbon chain of HOU-Cn, the permeability and conductivity cell membrane increased, which may result in the death of bacteria.3 The effect of HOU-Cn on the fatty acid synthase activity of Bacillus subtilisFatty acid synthase in Bacillus subtilis was a multi-enzyme complex, which formed by seven kinds of enzymes and an acryl carrier protein (ACP). Enzymological results displayed that HOU-Cn could inhibit the activity of FASâ…¡obviously. The inhibition of HOU-Cn to FASâ…¡increased with the elongation of carbon chain of HOU-Cn.HOU-C12 showed the highest inhibition.4 The effect of HOU-Cn on the content of fatty acids of B. subtilisFatty acids are important components in cell membrane, which are composed of saturated and unsaturated ones. Unsaturated fatty acids maintain the relative mobility of cell membrane. According to present results, HOU-Cn changed the proportion of fatty acids in B.subtilis,but also increased the relative content of saturated fatty acids and declined that of the unsaturated fatty acids.As a result, the rigidity of the cell membrane increased and the flexibility weakened. Therefore, the cells become more disruptive. With the elongation of carbon chain of HOU-Cn, the content of saturated fatty acid such as palmitic acid (C16:0), stearic acid (C18:0) and unsaturated fatty acid such as oleic acid (C18:1n-9) and linolenic acid (C18:3n-3) decreased gradually, but the relative proportion of saturated fatty acids has increased, unsaturated fatty acids reduce. Thereby, membrane rigidity of B.subtilis increased, viscosity decreased, and the cell membrane became easy to break.According to present results, HOU-Cn could weaken the supply of nutrients by destroying the glycocalyx and decreasing the fatty acid synthesis, and increase the permeability of membrane by increasing the fluidity of membrane, and change the percentage of saturated and unsaturated fatty acids by inhibiting FASII activity. Consequently, HOU-Cn showed a strong antibacterial activity against G+-bacteria by a multi-target antibacterial mechanism...