Functional Study Of Escherichia Coli K1 Pathogenicity Island GimA In Neonatal Meningitis

PurposeBacterial meningitis is a serious and sometimes fatal infection affecting the central nervous system (CNS). Despite great advances in antimicrobial chemotherapy and supportive care, bacterial meningitis remains an important cause of mortality and morbidity. The survivors often accompany with the hearing lose, blindness, epileptic, mental retardation and dyspraxia. A better understanding of the underlying pathogenic mechanisms of this disease may contribute to clinical intervention.Escherichia coli (E. coli) K1 is the most common gram-negative organism causing meningitis during the neonatal period. E. coli strains with the K1 capsular polysaccharide are the predominant isolates (about 80%) from neonatal E. coli meningitis. Evidences showed that most cases of E. coli meningitis occur as a result of hematogenous spread. Entry of circulating E. coli into the CNS requires passage across the blood-brain barrier (BBB), which is mainly composed of brain microvascular endothelial cells. To date, several genetic determinants contributing to bacterial crossing the BBB have been identified in E. coli K1, such as fimH, OmpA, ibeA, ibeB, CNF1, etc. Besides, studies showed that reorganization of the actins cytoskeleton and intracellular signaling such as PI3K,PKB,PKC,RhoGTPase and FAK in human brain microvascular endothelial cells (HBMECs) are involved in E. coli K1 invasion of HBMECs. However, the mechanism of these genetic determinants for E. coli K1 across the BBB is not clear.In our previous studies, a pathogenicity island GimA (genetic island of meningitic E. coli containing ibeA) (Fig.1) was identified from the genomic DNA library of E. coli K1 strain RS218 (018:K1:H7),a cerebrospinal fluid (CSF) isolate from a neonate with meningitis. The data suggested that GimA may contribute to E. coli invasion of the BBB. The entire block of 20.3 kb GimA sequences does not share significant homology with genome sequences of nonpathogenic E. coli K12. DNA sequence analysis showed that GimA consists of four operons, (1) PtnIPKC (GimA1), including ptnI,ptnP,ptnK,ptnC; (2) CglDTEC (GimA2), including cglD,cglT,cglE,cglC; (3) GcxKRCI (GimA3), including gcxK,gcxR,gcxC,gcxI; (4) IbeRAT (GimA4), including ibeR,ibeA,ibeT. The role of ibeA has been identified and it is involved in the invasion of HBMECs. Blast analysis results showed that ptnI in GimA1 share significant sequence homology with phosphoenolpyruvate-protein phosphoryltransferase; CglD in GimA2 is a putative glycerol dehydrogenase (GLDH), showing high homology to the paralogue in E. coli K12 strains (56%) and GLDH in other bacteria (up to 62%). GcxC in GimA3 share significant sequence homology with glycoxylate carboligase (up to 67%); one of the major functions of the operon GimA4 is being responsible for E. coli Kl invasion of host cells.IbeT and ibeR share few homology with other sequences and they are located at the same operon with ibeA, so they may be contributed to E.coli K1 invasion of HBMECs. Explore the role of these genes in E. coli K1 may be contributed to understand the mechanism of bacterial meningitis. Therefore, we carried our research to study the role of cglD in GimA2 and ibeT in GimA4 in the development of meningitis. Methods1. Functional study of Escherichia coti K1 pathogenicity island gene cglD(1) Bacterial cultureBacterial strains were grown in LB medium; before bacterial invasion assay, E44 were grown in BHI medium.(2) Cell cultureHBMECs were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 10% Nu serum at 37℃in a 5% CO2,100% air humidified atmosphere.(3) Construction of isogenic in-frame deletion mutant strainPlasmid pCVD-△cglD was introduced into E44 by plate mating. E44:△cglD were constructed by bacterial homologous exchange.(4) Complementation analysisORF of cglD gene was cloned into pFN476 vector and were transformed with pGP1-2 together into E44:△cglD.(5) Bacterial adhesion and invasion assaysBacterial adhesion assays:bacteria (10'CFU/well) were added to confluent monolayers of HBMECs, bacteria were collected after 1.5h and results were expressed as relative adhesion.Bacterial invasion assays:bacteria (107 CFU/well) were added to confluent monolayer of HBMECs for 1.5h. The number of intracellular bacteria was determined after the extracellular bacteria were killed, results were expressed as relative invasion.(6) Median lethal dose (LD50) and survival analysisLD50 was determined by Bliss method and survival analysis were determined by Kaplan-Meier survival curve.(7) Experimental rat model of neonatal meningitisBacteremia rat model was established and E. coli model of neonatal meningitis was a result of hematogenous spread.(8) White blood cell (WBC) count, protein and lactate levels in CSFWBC count was determined by hemocytometer. The concentrations of protein and lactate in CSF were measured by BCA Protein Assay Kit and Lactate Assay Kit.(9) Histological analysis(10) Polymorphonuclear neutrophils (PMNs) transendothelial migration assayFresh human PMNs were isolated from human peripheral blood. Migrated PMNs were counted in a hemacytometer.(11) Recombinant protein expression and purificationRecombinant plasmid was expressed in BL21 (DE3) and GST-CglD was purified by affinity chromatography. (12) Glycerol dehydrogenase (GLDH) assayThe activity of glycerol dehydrogenase was determined by colorimetric methods.2. Functional study of Escherichia coli Kl pathogenicity island gene ibeT(1) Bacterial cultureBacterial strains were grown in LB medium, before bacterial invasion assay, E44 were grown in BHI medium.(2) Cell cultureHBMECs were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 10% Nu serum.(3) Construction of isogenic in-frame deletion mutant strainPlasmid pCVD-△ibeT was introduced into E44 by plate mating. E44:△ibeT were constructed by bacterial homologous exchange.(4) Complementation analysisORF of ibeT gene was cloned into pFN476 vector and were transformed with pGPl-2 together into E44:△ibeT.(5) Bacterial adhesion and invasion assaysBacterial adhesion assays:bacteria (10'CFU/well) were added to confluent monolayers of HBMECs, bacteria were collected after 1.5h and results were expressed as relative adhesion.Bacterial invasion assays:bacteria (107 CFU/well) were added to confluent monolayer of HBMECs for 1.5h. The number of intracellular bacteria was determined after the extracellular bacteria were killed, and the results were expressed as relative invasion.(6) Bacterial survival in HBMECs assay Bacteria (107 CFU/well) were added to confluent monolayer of HBMECs for 1.5h, and intracellular bacteria were determined after the extracellular bacteria were killed at the indicated time, and results were expressed with bacterial CFU.(7) Bacterial grow curvesBacterial grown curves were determined by the absorbance values at A600nm.(8) Immunofluorescent technique(9) Transmission electron microscope (TEM)The embedding block were cut into 70-80nm thick, stained, and were scanned with TEM.(10) Determine the cytoplasmic buffering capacity of the bacteriaCytoplasmic buffering capacity was calculated by the difference between total buffering capacity and extracellular buffering capacity.Results1. The role of Escherichia coli K1 pathogenicity island gene cglD in experimental neonatal meningitis(1) Isogenic in-frame deletion of cglD gene in E. coli Kl.(2) CglD deletion in E. coli Kl leads to prolonged survival of the neonatal rats in experimental meningitis, without affecting the bacterial penetration through the BBB.(3) CglD deletion in E. coli Kl alleviated CSF change in the neonatal rats with experimental meningitis.(4) CglD deletion in E. coli K1 results in decreased neutrophils infiltration in the meninges and the cerebral cortex in experimental E. coli meningitis.(5) CglD protein has the enzymatic activity of glycerol dehydrogenase.2. IbeT, a Escherichia coli K1 pathogenicity island gene, is essential for escape from the lysosomes in human brain microvascular endothelial cells(1) Isogenic in-frame deletion of ibeT gene in E. coli K1.(2) IbeT deletion in E. coli K1 affects the bacterial invasion into HBMECs.(3) Deletion of ibeT gene inhibited the intracellular grown of E. coli K1 in HBMECs.(4) The E44:△ibeT mutant failed to escape the lysosomes of HBMECs(5) The E44:△ibeT mutant failed to escape from the ECV into the cytoplasm of HBMECs.(6) The cytoplasmic buffering capacity of E44:△ibeT mutant decreased.ConclusionCglD as a virulence factor for E. coli K1 contributed to the in vivo virulence in the bacterial meningitis and play an important role in the development of neonatal meningitis. It is not involved in bacterial across the BBB, but it is has the activity of glycerol dehydrogenase.The expression of ibeT in Escherichia coli Kl contributed to ECV membrane degradation and subsequent escape from lysosomes into the cytoplasm for replication after Escherichia coli K1 invasion into HBMECs.