Engineering a CRISPR interference system targeting AcrAB-TolC efflux pump to prevent multidrug resistance development in Escherichia coli.
Xiulin Wan, Qingyang Li, Rikke Heidemann Olsen, Hecheng Meng, Zhigang Zhang, Junlin Wang, Hanyu Zheng, Lili Li, Lei Shi
Author Information
Xiulin Wan: Institute of Food Safety and Nutrition, Jinan University, Guangzhou 510632, China.
Qingyang Li: College of Life Sciences, Hebei Normal University, Shijiazhuang 050024, China.
Rikke Heidemann Olsen: Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.
Hecheng Meng: School of Food Science and Engineering, South China University of Technology, Guangzhou, Guangdong, China.
Zhigang Zhang: State Key Laboratory of Food Safety Technology for Meat Products, Xiamen Yinxiang Group, Xiamen, China.
Junlin Wang: Institute of Food Safety and Nutrition, Jinan University, Guangzhou 510632, China.
Hanyu Zheng: Institute of Food Safety and Nutrition, Jinan University, Guangzhou 510632, China.
Lili Li: Institute of Food Safety and Nutrition, Jinan University, Guangzhou 510632, China.
Lei Shi: Institute of Food Safety and Nutrition, Jinan University, Guangzhou 510632, China.
OBJECTIVES: We engineered a CRISPR interference (CRISPRi) system targeting the AcrAB-TolC efflux pump to prevent MDR development in Escherichia coli. METHODS: Nine specific single-guide RNAs (sgRNAs) were designed to target the components of the AcrAB-TolC efflux pump, namely AcrA, AcrB and TolC. A total of thirteen CRISPRi recombinant plasmids were constructed with single or clustered sgRNAs. The transcriptional levels of the target genes, MICs of multiple antibiotics and biofilm formation in each CRISPRi strain were tested. RESULTS: The CRISPRi system expressing sgRNA clusters targeting acrB and tolC simultaneously exhibited the highest inhibitory effect on AcrAB-TolC efflux pump activity in E. coli HB101, with 78.3%, 90.0% and 65.4% inhibition rates on the transcriptional levels of acrA, acrB and tolC, respectively. The CRISPRi system resulted in ∼2-, ∼8- and 16-fold increased susceptibility to rifampicin, erythromycin and tetracycline, respectively. In addition, the constructed CRISPRi system reduced biofilm formation with inhibition rates in the range of 11.2% to 58.2%. CONCLUSIONS: To the best of our knowledge, this is the first report on the construction of an inducible CRISPRi system targeting the AcrAB-TolC efflux pump to prevent MDR development in E. coli. This study provides insights for future regulation and manipulation of AcrAB-TolC activity and bacterial MDR by a CRISPRi system.
Grants
2022A1515011685/Natural Science Foundation of Guangdong Province
32001796/National Natural Science Foundation of China
202002030630020049/Basic Research Project of Guangzhou
MeSH Term
Anti-Bacterial Agents
Bacterial Outer Membrane Proteins
Carrier Proteins
Clustered Regularly Interspaced Short Palindromic Repeats
