Department of Infectious Diseases and the Key Lab of Endogenous Infection, Shenzhen Nanshan People's Hospital and The 6th Affiliated Hospital of Shenzhen University Health Science Center, Shenzhen, 518052, China.
Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Department of Medical Microbiology and Parasitology, School of Basic Medical Science and Institutes of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai, 200032, China.
BMC Microbiol. 2022 Jan 7;22(1):16. doi: 10.1186/s12866-021-02423-8.
Previous reports have demonstrated two thiazolidione derivatives (H2-60 and H2-81) can robustly inhibit the planktonic growth and biofilm formation of S. epidermidis and S. aureus by targeting the histidine kinase YycG. Whereas the antibacterial and anti-biofilm activity of these two thiazolidione derivatives (H2-60 and H2-81) against Enterococcus faecium remains elusive. Here, the pET28a-YycG recombinant plasmid were in vitro expressed in E. coli competent cell BL21 (DE3) and induced to express YycG' protein (conding HisKA and HATPase_c domain) by 0.5 mM IPTG and was purified by Ni - NTA agarose and then for the autophosphorylation test. Antimicrobial testing and time-killing assay were also be determined. Anti-biofilm activity of two derivatives with sub-MIC concentration towards positive biofilm producers of clinical E. faecium were detected using polystyrene microtiter plate and CLSM.
The MICs of H2-60 and H2-81 in the clinical isolates of E. faecium were in the range from 3.125 mg/L to 25 mg/L. Moreover, either H2-60 or H2-81 showed the excellent bactericidal activity against E. faecium with monotherapy or its combination with daptomycin by time-killing assay. E. faecium planktonic cells can be decreased by H2-60 or H2-81 for more than 3 × log10 CFU/mL after 24 h treatment when combined with daptomycin. Furthermore, over 90% of E. faecium biofilm formation could markedly be inhibited by H2-60 and H2-81 at 1/4 × MIC value. In addition, the frequency of the eradicated viable cells embedded in mature biofilm were evaluated by the confocal laser microscopy, suggesting that of H2-60 combined with ampicillin or daptomycin was significantly high when compared with single treatment (78.17 and 74.48% vs. 41.59%, respectively, P < 0.01).
These two thiazolidione derivatives (H2-60 and H2-81) could directly impact the kinase phosphoration activity of YycG of E. faecium. H2-60 combined with daptomycin exhibit the excellent antibacterial and anti-biofilm activity against E. faecium by targeting YycG.
先前的研究报告表明，两种噻唑烷二酮衍生物（H2-60 和 H2-81）可以通过靶向组氨酸激酶 YycG 来有效抑制表皮葡萄球菌和金黄色葡萄球菌的浮游生长和生物膜形成。然而，这两种噻唑烷二酮衍生物（H2-60 和 H2-81）对粪肠球菌的抗菌和抗生物膜活性仍不清楚。在这里，通过体外表达 pET28a-YycG 重组质粒，在大肠杆菌感受态细胞 BL21（DE3）中表达 YycG'蛋白（编码 HisKA 和 HATPase_c 结构域），并用 0.5 mM IPTG 诱导表达，并通过 Ni-NTA 琼脂糖进行纯化，然后进行自动磷酸化测试。还测定了抗菌试验和杀菌试验。采用聚苯乙烯微量滴定板和 CLSM 检测两种衍生物在亚最小抑菌浓度下对临床粪肠球菌阳性生物膜生产者的抗生物膜活性。
H2-60 和 H2-81 在粪肠球菌临床分离株中的 MIC 值在 3.125 mg/L 至 25 mg/L 范围内。此外，时间杀伤试验显示，无论是 H2-60 还是 H2-81，与达托霉素联合使用时，对粪肠球菌均具有良好的杀菌活性，单药或联合用药。H2-60 或 H2-81 与达托霉素联合使用 24 小时后，粪肠球菌浮游细胞减少超过 3 log10 CFU/mL。此外，在 1/4 MIC 值时，H2-60 和 H2-81 可显著抑制超过 90%的粪肠球菌生物膜形成。此外，通过共聚焦激光显微镜评估嵌入成熟生物膜中的根除活细胞的频率，与单一治疗相比，H2-60 联合氨苄西林或达托霉素的效果明显更高（分别为 78.17%和 74.48% vs. 41.59%，P<0.01）。
这两种噻唑烷二酮衍生物（H2-60 和 H2-81）可直接影响粪肠球菌 YycG 的激酶磷酸化活性。通过靶向 YycG，H2-60 与达托霉素联合使用对粪肠球菌具有良好的抗菌和抗生物膜活性。
