萎蔫酸铜离子络合物、镉离子络合物的抗结核活性及对结核分枝杆菌基因表达的影响

The anti-tuberculosis activity of metal complexes of fusaric acid and their effect on gene expressions of M.tuberculosis

目的研究海洋微生物代谢产物萎蔫酸金属铜离子络合物与镉离子络合物体外抗结核活性,以及对Mtb H37Rv基因表达的影响。方法应用纸片扩散法(Kind-Bauer,K-B法)初步鉴定萎蔫酸铜离子络合物、萎蔫酸镉离子络合物的抗结核活性,重复实验3次;绝对浓度间接法测定萎蔫酸铜离子络合物、萎蔫酸镉离子络合物的最低抑菌浓度(minimum inhibitory concentration,MIC),重复实验确定更准确的MIC;Mtb cDNA芯片检测萎蔫酸铜离子络合物、萎蔫酸镉离子络合物处理Mtb组与溶剂对照处理Mtb组表达差异的基因,重复实验一次。结果萎蔫酸铜离子络合物、萎蔫酸镉离子络合物在卡介苗(BCG)平板抑菌圈直径分别为(30.4±0.6)mm、(30.0±0.8)mm。萎蔫酸铜离子络合物抗Mtb H37Rv的MIC为10μg/ml;对耐多药结核分枝杆菌,萎蔫酸镉离子络合物MIC为7.5μg/ml,低于S(MIC=25μg/ml)和RFP(MIC=25μg/ml);对Mtb临床分离株(0907961,耐S和EMB),萎蔫酸铜离子络合物的MIC(10μg/ml)低于S(S的MIC为20μg/ml),萎蔫酸镉离子络合物的MIC(5μg/ml)均低于S(MIC=20μg/ml)、EMB(MIC=6.4μg/ml)。Mtb cDNA芯片检测发现萎蔫酸铜离子络合物处理组与溶剂对照组比较,有差异表达的基因总数为23条,其中已知功能基因有10条;萎蔫酸镉离子络合物处理组与溶剂对照组比较,有差异表达的基因总数为28条,其中已知功能基因有11条。这些存在差异的基因功能涉及核苷酸、脂质、能量、辅酶、氨基酸代谢,DNA的复制、转录、翻译、翻译后修饰以及细胞膜的生物合成等。结论萎蔫酸铜离子络合物与萎蔫酸镉离子络合物具有较好的体外抗结核活性,尤其对部分耐药菌株的抑制作用甚至优于抗结核一线用药,可作为抗结核药物的前导化合物;运用基因芯片所探究出的差异表达的基因为进一步深入研究其抗菌作用机制提供了一定的实验依据。

Objective To observe in vitro anti-tuberculosis activity of copper ion complex and cadmium ion complex of fusaric acid, and their effects on the gene expressions of M. tuberculosis （.Mtb） H37Rv. Methods The anti-tuberculosis activities of copper ion complex and cadmium ion complex of fusaric acid were determined for three times by Kind-Bauer method, and their minimum inhibitory concentrations （MICs） were detected twice by the abso- lute concentration method. The differences of the gene expressions of Mtb H37Rv between the compound group and non-compound control group were measured twice with DNA chips. Results The antibacterial diameters of cadmium complex and cupric complex of fusaric acid on the DCG plates were （30. 0±0. 8） mm and （30. 4±0. 6） mm, respectively. The MIC of cupric complex of fusarie acid on Mtb H37Rv was 10μg/ml. The MIC of cadmium complex of fusaric acid on multi-drug-resistant Mtb was 7.5 μg/ml, which was lower than that of streptomycin （S, MIC= 25 μg/ml） and rifampicin （RFP, MIC= 25 μg/ml）. The MIC of cupric complex of fusarie acid on Mtb clinical isolate（No. 0907961, resistant to S and EMB） was 10μg/ml, which was lower than that of S （MIC= 20 bμg/ml） ; and the MIC of cadmium complex of fusaric acid was 5μg/ml, which was lower than that of S （MIC=20μg/ml） and EMB （MIC= 6.4/~g/ml）. Of 3875 genes tested by the DNA array, 23 genes （10 known functional genes） were significantly dif- ferent in copper ion complex group, and 28 genes （11 known functional genes） in cadmium ion complex group com- pared with the control groups. The functions of these differential genes were involved in nucleotide, lipid, energy, eoenzyme, amino acid metabolism, DNA replication, transcription, translation, post-translational modification, bio- synthesis of cell membranes etc. Conclusion Metal complexes of fusaric acid appeared considerable in vitro anti tuberculosis activity, especially inhibitory activities of the compounds on some drug-resistant Mtb strains appeared higher than that of the first-line agents. Therefore, they could be used as anti-tuberculosis lead compounds. The genes with differential expressions showed by the DNA chip provide a valuable foundation for further study on their antibacterial mechanism.