Study Of Glucose Mediated Cyclic AMP-CRP Regulating The Morphology Mechanism Of Klebsiella Pneumoniae

Objective: Klebsiella pneumoniae(KP)is a rod-shaped gram-negative enterobacteriaceae that can cause pneumonia,empyema,urinary tract infections,or bacteremia.Among them,K1 / K2 serotype can cause acquired liver abscess and invasion infections such as endophthalmitis.Many studies had proved that diabetes was a high risk factor for K.pneumoniae invasive infection.How does the diabetic environment affect K.pneumoniae? Bacterial morphology distinguishes bacterial species and regulates bacterial attachment and pathogenicity.Bacteria can modify their morphology in response to their host environmental stimuli or different growth conditions.Nutritional status is a factor favoring bacterial shape modification that affects the nutrient acquisition.The effect of high glucose in diabetic patients on the cell morphology of K.pneumoniae NTUH-2044,a capsular serotype K1 strain with hypermucoviscosity phenotype,is still unknown.Methods: The mice model of type II diabetes mellitus(T 2 DM)was established.K.pneumoniae NTHU-2044 strain(or digestive tract)was infected intraperitoneally.The influence of diabetes on the pathogenicity and the bacterial load of liver were analyzed.Meanwhile,the morphology of K.pneumoniae in liver was observed by liver homogenate gram stain.In order to analyze the mechanism of morphological changes of K.pneumoniae,the crp gene deletion mutant and the complementary stains were constructed.The morphological changes of WT,crp deletion mutant strain(△crp)and crp complement strain(Ccrp)were analyzed by SEM.Then the proteomic changes of WT and △crp were analyzed,and the genes related to the division and cell wall synthesis of bacteria that may be regulated by CRP were seleeted out.The expression of these genes in high glucose environment was analyzed by q RT-PCR.The strains were expressed the gene whose expression lever was increased in high glucose environment were constructed and the morphology of them were observed.On this basis,the regulatory mechanism of CRP to the related genes was studied by Lac-Z fusion experiment and Electrophoretic mobility shift assay(EMSA).Results: The pathogenicity of K.pneumoniae in type II diabetes model mice was significantly increased compared to control.The morphology of K.pneumoniae in the liver of diabetic mice became shorter and rounder,no longer rod-shaped(1.387 ± 0.089 μm vs 2.506 ± 0.131 μm,P < 0.01).The morphology of K.pneumoniae also changed in high glucose medium in vitro.Compared to WT,△crp strain became short and round(0.981 ± 0.057 μm vs 2.415 ± 0.066 μm,P ≤ 0.001),while C-crp regained rod-shaped morphology.High glucose concentration in vivo and in vitro can shorten the morphology of K.pneumoniae,and the length of △crp is significantly shorter than that of WT.These results show that the high glucose environment can change the bacterial morphology by regulation the activity of c AMP-CRP complex.Comparative proteomic analysis showed that in Δcrp,the expression of 10 proteins involved in cell division or cell wall biosynthesis was increased.Five of them(omp A,tol B,ybg C,fts I,rcs F)were selected to verify their expression in high glucose environment.The results showed that the expression levels of rcs F,tol B and fts I increased significantly in LB medium containing 12 m M glucose.And the overexpression of tol B or rcs F shortened the length of bacteria,similar to △crp.Lac Z and EMSA experiments showed that CRP directly and negatively regulated the transcription of tol B and rcs F by binding to promoter region.Conclusion:(1)The high glucose environment can change the bacterial morphology;(2)CRP was involved in the regulation of bacterial morphology;(3)The high glucose environment regulates the activity of c AMP-CRP,and affects the bacterial morphology by directly negatively regulating genes tol B and rcs F,which change bacterial division and bacterial morphology.In this study,we analyzed the effection of high glucose environment on the morphology of Klebsiella pneumoniae and its possible regulatory mechanism.This study first proved the role and regulation mechanism of CRP in the alteration in cell morphology during infection in DM patiants.