A Screening Model of Antibacterial Agents Based on <i>Escherichia coli</i> Cell-Division Protein
Pathogenic <i>Escherichia coli</i> cannot be killed by most antibiotics (including colistin, a last-resort drug) due to the rapid development of antibiotic resistance. A highly conserved prokaryotic mitotic protein, filamenting temperature-sensitive protein Z (FtsZ) with GTPase activity,...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-04-01
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| Series: | Applied Sciences |
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| Online Access: | https://www.mdpi.com/2076-3417/13/7/4493 |
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| author | Qiuyu Fan Jianwen Wu Bolin Xi Chunxiao Li Xiumin Wang Huanrong Li |
| author_facet | Qiuyu Fan Jianwen Wu Bolin Xi Chunxiao Li Xiumin Wang Huanrong Li |
| author_sort | Qiuyu Fan |
| collection | DOAJ |
| description | Pathogenic <i>Escherichia coli</i> cannot be killed by most antibiotics (including colistin, a last-resort drug) due to the rapid development of antibiotic resistance. A highly conserved prokaryotic mitotic protein, filamenting temperature-sensitive protein Z (FtsZ) with GTPase activity, plays a key role in cell division and has become a promising target for screening novel antibacterial agents. In this study, the amplified <i>ftsZ</i> gene was inserted into cloning/expression vectors and recombinantly produced in <i>E. coli</i>; the recombinant FtsZ protein was purified by the Ni<sup>2+</sup>-NTA affinity column and then was used to screen for natural antibacterial agents. The results showed that the <i>ftsZ</i> gene with a size of 1170 bp was successfully amplified from <i>E. coli</i> and inserted into the pET-28a expression vector. After induction with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), FtsZ was expressed in <i>E. coli</i> BL21 as inclusion bodies. After purification, the recombinant FtsZ protein showed GTPase activity. The highest GTPase activity (0.998 nmol/mL/min) of FtsZ was observed at a GTP concentration of 1.25 mM. Several alkaloids were screened by a constructed model of FtsZ inhibitors. Sanguinarine chloride exhibited higher antibacterial activity against <i>E. coli</i> and <i>Salmonella enteritidis</i> (with minimum inhibitory concentrations (MICs) of 0.04–0.16 mg/mL and minimum bactericidal concentrations (MBCs) of 0.16–0.32 mg/mL) than tetrandrine (0.16–0.32 mg/mL) and berberine hydrochloride (0.32–0.64 mg/mL). Berberine hydrochloride prevented FtsZ polymerization in a concentration-dependent manner and bound to FtsZ protein by hydrogen bonding interaction. This study suggested that the FtsZ-based <i>E. coli</i> screening model could be exploited for the development of novel antibacterial agents for clinical applications. |
| first_indexed | 2024-03-11T05:42:15Z |
| format | Article |
| id | doaj.art-cf0e2078181f4312a29d539fe7372160 |
| institution | Directory Open Access Journal |
| issn | 2076-3417 |
| language | English |
| last_indexed | 2024-03-11T05:42:15Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Applied Sciences |
| spelling | doaj.art-cf0e2078181f4312a29d539fe73721602023-11-17T16:21:09ZengMDPI AGApplied Sciences2076-34172023-04-01137449310.3390/app13074493A Screening Model of Antibacterial Agents Based on <i>Escherichia coli</i> Cell-Division ProteinQiuyu Fan0Jianwen Wu1Bolin Xi2Chunxiao Li3Xiumin Wang4Huanrong Li5College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, ChinaCollege of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, ChinaSchool of Life Sciences, Jilin University, Changchun 130012, ChinaInstitute of Feed Research, Chinese Academy of Agricultural Sciences, Beijing 100081, ChinaInstitute of Feed Research, Chinese Academy of Agricultural Sciences, Beijing 100081, ChinaCollege of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, ChinaPathogenic <i>Escherichia coli</i> cannot be killed by most antibiotics (including colistin, a last-resort drug) due to the rapid development of antibiotic resistance. A highly conserved prokaryotic mitotic protein, filamenting temperature-sensitive protein Z (FtsZ) with GTPase activity, plays a key role in cell division and has become a promising target for screening novel antibacterial agents. In this study, the amplified <i>ftsZ</i> gene was inserted into cloning/expression vectors and recombinantly produced in <i>E. coli</i>; the recombinant FtsZ protein was purified by the Ni<sup>2+</sup>-NTA affinity column and then was used to screen for natural antibacterial agents. The results showed that the <i>ftsZ</i> gene with a size of 1170 bp was successfully amplified from <i>E. coli</i> and inserted into the pET-28a expression vector. After induction with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), FtsZ was expressed in <i>E. coli</i> BL21 as inclusion bodies. After purification, the recombinant FtsZ protein showed GTPase activity. The highest GTPase activity (0.998 nmol/mL/min) of FtsZ was observed at a GTP concentration of 1.25 mM. Several alkaloids were screened by a constructed model of FtsZ inhibitors. Sanguinarine chloride exhibited higher antibacterial activity against <i>E. coli</i> and <i>Salmonella enteritidis</i> (with minimum inhibitory concentrations (MICs) of 0.04–0.16 mg/mL and minimum bactericidal concentrations (MBCs) of 0.16–0.32 mg/mL) than tetrandrine (0.16–0.32 mg/mL) and berberine hydrochloride (0.32–0.64 mg/mL). Berberine hydrochloride prevented FtsZ polymerization in a concentration-dependent manner and bound to FtsZ protein by hydrogen bonding interaction. This study suggested that the FtsZ-based <i>E. coli</i> screening model could be exploited for the development of novel antibacterial agents for clinical applications.https://www.mdpi.com/2076-3417/13/7/4493FtsZexpressionpurificationGTPasealkaloidantibacterial activity |
| spellingShingle | Qiuyu Fan Jianwen Wu Bolin Xi Chunxiao Li Xiumin Wang Huanrong Li A Screening Model of Antibacterial Agents Based on <i>Escherichia coli</i> Cell-Division Protein Applied Sciences FtsZ expression purification GTPase alkaloid antibacterial activity |
| title | A Screening Model of Antibacterial Agents Based on <i>Escherichia coli</i> Cell-Division Protein |
| title_full | A Screening Model of Antibacterial Agents Based on <i>Escherichia coli</i> Cell-Division Protein |
| title_fullStr | A Screening Model of Antibacterial Agents Based on <i>Escherichia coli</i> Cell-Division Protein |
| title_full_unstemmed | A Screening Model of Antibacterial Agents Based on <i>Escherichia coli</i> Cell-Division Protein |
| title_short | A Screening Model of Antibacterial Agents Based on <i>Escherichia coli</i> Cell-Division Protein |
| title_sort | screening model of antibacterial agents based on i escherichia coli i cell division protein |
| topic | FtsZ expression purification GTPase alkaloid antibacterial activity |
| url | https://www.mdpi.com/2076-3417/13/7/4493 |
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