Mechanism of transcriptional regulator CRP in regulating carbapenem-resistant Klebsiella pneumoniae entC
10.3760/cma.j.cn112309-20230530-00145
- VernacularTitle:转录调控子CRP对碳青霉烯类耐药肺炎克雷伯菌 entC的调控机制
- Author:
Jiandie BI
1
;
Qiuyue HE
;
Shumin LIU
;
Min NIU
;
Kai YANG
;
Yan DU
Author Information
1. 昆明医科大学第一附属医院医学检验科，云南省检验医学重点实验室，云南省医学检验临床医学研究中心，昆明 650032
- Keywords:
Carbapenem-resistant Klebsiella pneumoniae;
CRP;
Enterobactin;
Transcriptional regulation
- From:
Chinese Journal of Microbiology and Immunology
2023;43(10):733-739
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the mechanism of cyclic AMP receptor protein (CRP) in regulating the siderophore enterobactin-related gene entC of carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods:A mutant strain with crp gene deletion strain (Δ crp) and a complementary strain (c-Δ crp) were constructed using CRKP-27 as the wild-type strain. The influence of CRP on the secretion of siderophore by CRKP was analyzed by chrome azurol sulfonate (CAS) quantitative assay. RT-qPCR and lacZ reporter gene fusion assay were used to detect the regulatory effect of CRP on entC gene expression and its promoter. Electric mobility shift assay (EMSA) was performed to detect the binding of CRP to the entC promoter region and the binding sequence was analyzed by DNase Ⅰ footprinting assay. Results:The Δ crp and c-Δ crp strains were successfully constructed. Compared with the wild-type and c-Δ crp strains, the Δ crp strain could secrete more siderophore under both normal and iron-deficient conditions, but the difference was statistically significant only under normal condition ( P<0.05). The relative expression of entC gene at mRNA level was significantly lower in the Δ crp strain than that in the wild-type and c-Δ crp strains under both normal and iron-deficient conditions (both P<0.05). The promoter of entC gene in the Δ crp strain was less active than that in the wild-type and c-Δ crp strains under both normal and iron-deficient conditions (both P<0.05). EMSA showed that with the increase of CRP protein, the distance of entC probe from the positive pole was shortened and blocked. DNase Ⅰ footprinting assay further identified the specific binding site of the entC promoter region to CRP as 5′-AAGGTGATAAATGCGTCTCATTTTCAA-3′. Conclusions:The CRP protein in CRKP could specifically bind to the entC promoter region and directly promote its expression at transcriptional level.
