Identification Of RcsAB Transcriptional Profile In Klebsiella Pneumoniae By Microarray

Objective: Klebsiella pneumoniae(K.pneumoniae)is an important opportunistic pathogen in clinical that cause pneumonia,bacteremia,liver abscess,urinary tract infection and other clinical disease.Rcs phosphorelay system is a two-component signal transduction system in bacteria which could regulate the virulence gene transcription in some Enterobacter.In this study,microarray technology was used to screen the overall genes regulated by RcsA?RcsB and RcsAB in genome-Wide in order to identificate the transcriptional profile of RcsAB in Klebsiella pneumoniae.The results of the bioinformatics analysis of these target genes provide the evidence to further understand the effect of RcsAB on the virulence of Klebsiella pneumoniae.Methods: The mutants Kp-?rcsA?Kp-?rcs B?Kp-?rcs AB were generated.Total RNA of the strains were isolated and labeled,ant then cDNA was synthesis.Chip hybridization was finished and the microarray results were scanned.Statistical and bioinformatic analysis was used to the raw data and got differentially expressed genes(DEGs).RT-qPCR and LacZ fusion assay were used to confirm the microarray results.Then,DEGs were sorted into functional categories based on clusters of orthologous groups(COGs).Results: 1.43 genes regulated by RcsA were selected,including 16 upregulated and 27 downregulated genes;a total of 223 genes regulated by RcsB,of which 91 upregulated and 132 were downregulated;210 genes independently regulated by RcsAB,of which 83 were upregulated genes and 127 were downregulated.3.RT-qPCR technology was used to verify the microarray results of the 14 randomly selected genes.By comparison,the consistency of the two experimental results was 92.86%.It is indicated that microarray data had high reliability.4.The LacZ fusion assay showed that the results of fadJ were in accordance with the RT-qPCR results.5.COG analysis indicated that DEGs regulated by RcsA were mainly related to inorganic ion transport and metabolism,carbohydrate transport and metabolism,lipid transport and metabolism and cell wall/membrane/envelope biogenesis;DEGs regulated by Rcs B were mainly related to energy production and conversion,carbohydrate transport and metabolism,cell wall/membrane/envelope biogenesis;DEGs regulated by Rcs AB were mainly related to carbohydrate transport and metabolism and cell wall/membrane/envelope biogenesis.6.GO analysis showed that DEGs regulated by RcsA participated in catalytic activity,cofactor binding,single-organism process;DEGs regulated by RcsB participated in catalytic activity,membrane-bounded organelle biosynthetic process,cytoplasmic part biosynthetic process and other physiological and biochemical processes;DEGs regulated by RcsAB participated in catalytic activity,carboxylic acid metabolic process,oxoacid metabolic process.Conclusions: In this study,microarray technology was used to screen target genes regulated by RcsA,RcsB and RcsAB,and the chip results were verified by RT-qPCR and LacZ fusion assay.COG and GO analysis were used to confirm the genes functions to provide foundation for studying the virulence mechanism of Klebsiella pneumoniae.Further,phenotypic experimental analysis of the differentially expressed genes could be carried out to help us better understand the influence of Rcs phosphorelay system on Klebsiella pneumoniae virulence,which may provide a theoretical basis for the prevention and treatment of Klebsiella pneumoniae infection in clinic.