Construction of BPSL1972 null mutation strain of Burkholderia pseudomallei and its biological characterization
- Author:
ZHU Qiao
;
TIAN Shen
;
WANG Yanshuang
- Publication Type:Journal Article
- Keywords:
Burkholderia pseudomallei;
efflux pump;
drug resistance
- From:
China Tropical Medicine
2024;24(6):644-
- CountryChina
- Language:Chinese
-
Abstract:
Abstract: Objective To study the biological function of the possible efflux pump gene BPSL1972 in Burkholderia pseudomallei (Bp), so as to improve the drug resistance mechanism of Bp. Methods BPSL1972 gene knockout strain and its complement strain were constructed by homologous recombination and electric shock transformation, respectively. BPSL1972 protein expression vector was constructed by enzyme digestion and enzyme-linked method. The BPSL1972-encoded protein was expressed using a His-tag protein expression kit and the expressed proteins were verified by Western Blot. The changes in drug sensitivity, growth, biofilm formation, and cytotoxicity were compared by microbroth dilution assay, Kirby-Bauer assay, turbidimetric assay, tube assay, crystal violet staining, and CCK-8 kit. The physicochemical properties of BPSL1972 were predicted by Protscale and Protparam online websites, the signal peptide of BPSL1972 was predicted by SignalP 5.0 online website, and the transmembrane domain and gene function of BPSL1972 were predicted by Smart online website. The 3D structure of BPSL1972-encoded protein and its homology were analyzed through Phyre 2 online website. Results The predictions suggested that the BPSL1972-encoded protein is a class of neutral-stable hydrophobic protein with a lipoprotein signal peptide and a transmembrane region, classified under the RND efflux pump system with two lipoprotein outer membrane efflux proteins, potentially mediating macrolide resistance. Its 3D modeling results show high homology to VceC and fusaric acid efflux protein, possibly mediating the efflux of ion activators. Sequencing results confirmed the successful construction of both the deletion mutant strain and complement strain. However, there were no significant differences in biological characteristics between the deletion mutant strain and the wild-type strain, and there was also no significant difference in resistance to antibiotics such as cephalosporins, quinolones, carbapenems, aminoglycosides, tetracyclines, and surfactants such as sodium deoxycholate. Conclusion BPSL1972 may be an auxiliary gene of LysR family, and this auxiliary gene is secondary, thus resulting in no functional changes after its deletion. Further research is needed to investigate whether it has resistance to other antibiotics such as fusaric acid and its specific replacement or auxiliary function.
- Full text:2025061809570479093.Construction of BPSL1972 null mutation strain of Burkholderia pseudomallei and its biological characterization.pdf
