The antibacterial activity and mechanism of ginkgolic acid C15:1
In this study, E. coli DH5?, E. coli O157: H7, P. putida KT2440, P. aeruginosa PAO1, R. solanacearum, Rhodococcus RHA1, S. thermophilus ND03, S. aureus and other common strains were used to study the antibacterial activity of GA (C15:1), and GA was found to have significant antibacterial activity against Gram-positive bacteria but little effect on the growth of Gram-negative bacteria. A relatively strong selective antibacterial mechanism of GA was observed. The MIC value of B. amyloliquefaciens SQR9 was the smallest among all of the tested Gram-positive bacteria. However, its MBC value (60 ?g mL?1) was the largest among all of the tested Gram-positive bacteria. These results might be caused by small amount of endospores that were generated when B. amyloliquefaciens SQR9 was cultured. Endospores had relatively strong resistance, and could withstand higher concentrations of GA without being killed. Therefore, B. amyloliquefaciens SQR9 had significantly higher MBC values than other Gram-positive bacteria. The antibacterial activity of GA has been reported these years. Himejima and Kubo [12] found that 2-hydroxy-6-(8-pentadecenyl) salicylic (another name of ginkgolic acid C15:1) showed lower MICs (about 10 ?g mL?1) against Gram-positive bacteria and higher MICs (100 ?g mL?1) against Gram-negative bacteria. Choi et al. [23] also showed that GA (C15:1) had significant antibacterial activity against 18 g-positive vancomycin-resistant. The results of the present study are consistent with above studies.
Additional studies on antibacterial mechanisms using GFP fluorescence-labeled Gram-positive bacteria B. amyloliquefaciens SQR9 and GFP-labeled Gram-negative bacteria E. coli DH5? and P. putida KT2440 showed that GA (C15:1) could significantly affect GFP fluorescence in the cells of Gram-positive B. amyloliquefaciens SQR9-gfp, whereas it had no significant effect on GFP fluorescence in the cells of Gram-negative bacteria E. coli DH5?-gfp and P. putida KT2440-gfp. The green fluorescent protein (GFP) has been widely used as a highly useful tool in the fluorescence studies of living cells, which is found in cell cytoplasm of jellyfish and is an extremely stable protein with 238 amino acids [25, 26]. The fluorescence produced by GFP was caused by its protein conformation. In general, as long as the protein conformation of GFP did not change, the fluorescence would not decay or disappear. Previous reports showed that GA and sumac acids, which had a similar structure, could affect the activity of numerous enzymes, including protein phosphatase, lipoxygenase and histone acetyltransferase [27–29]. In addition, GA affected in vivo regulation mechanism of small ubiquitin-related modifier (SUMO) and altered protein conformation, thereby affecting protein expression [30].
According to the above test, we suggested that the mechanism by which GA (C15:1) decayed GFP fluorescence was through conformation changes in the GFP protein. In addition, the mechanism by which GA promoted antibacterial activity against Gram-positive bacteria was through conformational changes of the proteins in the bacteria that inactivated the proteins and inhibited the growth of Gram-positive bacteria.
The results of crude cell lysate experiments showed that GFP fluorescence decay might be related to the interaction between GFP and GA. The GFP fluorescence in both Gram-negative and Gram-positive bacteria crude lysates was quenched by GA in a short period of time, which indicated that GFP fluorescence would be quenched as long as it had contact with GA and was not related to the microorganism tagged with GFP. Because the structure between Gram-negative and Gram-positive bacteria was similar and results showed that peptidoglycan in Gram-positive bacteria could not prevent GA (C15:1) from entering the cell, we suggested that the peptidoglycan structure of Gram-negative bacteria also could not block GA (C15:1) from entering the cell. In the protoplast experiment, a small amount of GA molecules could enter the cells after the peptidoglycan structure in E. coli cell wall was destroyed by lysozyme, which might be the result of the action of lysozyme. After the peptidoglycan structure was destroyed by lysozyme, pores might be present on the surface of the peptidoglycan layer that allowed GA molecules to pass through. However, only a small amount of GA molecules could enter the cells because the number of pores generated on the surface of the peptidoglycan layer was low, and the surface of Gram-negative bacteria was still covered by a large amount of lipids (including lipopolysaccharides and phospholipids), which could intercept a large amount of GA molecules. In order to further confirm lipid-soluble components in the cell wall of Gram-negative bacteria intercept the majority of GA molecules, the studies use high resolution electron microscopy to observe membrane change or other methods to study transport of GA through membrane will be carried out.
Some studies demonstrated that GA markedly inhibited the biofilm formation of S. mutans and Escherichia coli O157:H7, and disrupted biofilm integrity [24, 31]. Therefore, we speculate that the GA may affect the secondary metabolism of Gram-positive and Gram-negative bacteria. Due to the secondary metabolism of bacteria, such as the formation of biofilm, fluorescence formation and synthesis of antibiotics are regulated by quorum-sensing, further studies on this section will be investigated.