Exploration Of The Relationship Between The Transcriptional Regulator RcsAB And Capsule Polysaccharide-related Genes In Klebsiella Pneumoniae

Objective: Klebsiella pneumoniae(K.pneumoniae)is a common conditional pathogen causing nosocomial infections.The Rcs system is an important transcriptional regulatory system in K.pneumoniae,which is involved in the regulation of various virulence factors such as capsular polysaccharide.In this study,7 candidate genes related to capsular polysaccharide(rmpA,manB,wcaJ,wcaI,wcaG,gmd,magA)were selected from the NTUH-2044 genome.The purpose of our study was to investigate the transcriptional regulation relationship between RcsAB and the 7 candidate genes.Methods: Our laboratory has completed the screening of the target genes of RcsA,RcsB and RcsAB in the whole genome of NTUH-K2044 by using gene chip technology.On this basis,combined with literature review,a total of 7 candidate genes related to capsular polysaccharide(rmpA,manB,wcaJ,wcaI,wcaG,gmd,magA)were selected for our study.RT-qPCR and gene chip results were compared to preliminarily determinethe positive or negative regulatory relationship between the 7 candidate genes and the RcsA,RcsB and RcsAB.Then,the LacZ reporter gene fusion experiment was used to further determine the positive or negative regulatory relationship between RcsA,RcsB,RcsAB and the 7 candidate genes.EMSA was used to verify the regulatory relationship between RcsAB and the 7 candidate genes were direct or indirect regulation.Results: 1.RT-qPCR results showed that RcsB may positively regulated manB,wcaG and magA;RcsA and RcsB may positively regulated rmpA,wcaJ,wcaI and gmd.2.Because the results of RT-qPCR experiment and chip results were different,the LacZ gene fusion experiment was used for further verification.The results showed that RcsB positively regulated manB,wcaJ,wcaG,and magA;RcsA and RcsB positively regulated rmpA,wcaI,and gmd.3.For the EMSA results,no obvious binding bands were found between the 7 candidate genes and the RcsAB,indicating that the regulatory relationship were all indirect regulation.Conclusions: RT-qPCR and LacZ reporter gene fusion assay demonstrated that the transcriptional regulator RcsB may positively regulate the expression of manB,wcaJ,wcaG,and magA;RcsA and RcsB may positively regulate the expression of rmpA,wcaI,and gmd.It was proved by EMSA experiments that there may be an indirect regulatory relationship between RcsAB and these 7 capsular polysaccharide-related genes.