新生儿脑膜炎大肠杆菌毒力岛基因GimA的功能研究
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摘要
目的
新生儿细菌性脑膜炎是儿科严重感染性疾病之一,临床上尽管可以选用相应的抗生素治疗,但是病死率仍然得不到显著的改善。幸存者常伴有失听、失明、癫痫、智力和运动障碍后遗症。因此,探讨有效的新生儿细菌性脑膜炎的防治策略具有重要意义。
大肠杆菌(Escherichia coli, E. coli)是导致新生儿脑膜炎最常见的革兰氏阴性致病菌。从脑膜炎患儿脑脊液中分离的大肠杆菌中,80%以上均带有K1荚膜。现已清楚,E.coli K1通过血行播散入脑而引起脑膜炎。但是,血液中的E.coli K1如何穿过主要由脑微血管内皮细胞(brain microvascular endothelial cells, BMECs)组成的血脑屏障(blood brain barrier, BBB),迄今尚不清楚。借助于体外分离培养的人脑微血管内皮细胞(human brain microvascular endothelial cells, HBMECs)单层——血脑屏障模型,学者们先后鉴定了E. coli Kl中的参与侵袭HBMECs相关基因,如fimH, ompA, ibeA, ibeB, CNF1等,并对参与E.coli K1侵袭相关的细胞内信号转导通路如PI3K、PKB、PKC、RhoGTPase以及FAK等细胞骨架组装调控信号分子等进行了探讨。但已鉴定的这些E. coli K1穿过BBB的“决定因子”,特别是每个E. coli K1侵袭基因介导细菌侵袭HBMECs的功能差异、联系、及详细机制尚有待进一步研究。
我们在先前的研究中,从致新生儿脑膜炎E.coli K1株RS218(O18:K1:H7)中克隆鉴定了一个E.coli K1毒力岛基因GimA (genetic island of meningitic E.coli containing ibeA)(图1)。GimA全长20.3kb,仅存在于E.coli K1,其他非脑膜炎大肠杆菌中如E.coli K12均不含GimA序列。GimA含有四个操纵子,编码15个基因：(1)ptnIPKC (GimAl),包括ptnI、ptnP、ptnK、ptnC;(2)CglDTEC(GimA2),包括cglD、cglT、cglE、cglC;(3)GcxKRCI(GimA3),包括gcxK、gcxR、gcxC、gcxI; (4) IbeRAT(GimA4),包括ibeR、ibeA、ibeT。其中,ibeA基因的功能已得到初步研究,它在Kcoki K1侵袭HBMECs中起作用；其余14个基因的功能尚不清楚。GimA中推断的各基因编码蛋白与目前GeneBank库中功能已知蛋白之间的同源性高低程度分析结果提示：GimAl中ptnl与磷酸烯醇型丙酮酸磷酸化转移酶有很高的同源性；GimA2中cglD与甘油脱氢酶的同源性为62%;GimA3中gcxC与乙醛酸.醛连接酶的同源性高达67%;GimA4中含有E.coli K1侵袭HBMECs有关的ibeA,而其中的两个基因ibeT和ibeR尚未发现与其他蛋白有较高的同源性,鉴于这两个基因与ibeA同处于一个操纵子,故提示可能参与E.coli K1侵袭HBMECs.探讨这些基因在Ecoli Kl致脑膜炎中的作用,可以使我们更好的了解大肠杆菌性脑膜炎的发病机制。因此,我们选取了GimA2中的cglD基因和GimA4中的ibeT基因作为研究对象,展开了本工作。
方法
一、大肠杆菌毒力岛基因cglD的功能研究
(一)细菌培养
大肠杆菌以LB培养基培养,在行细胞侵袭试验前,用含有利福平的BHI培养基培养E44。
(二)细胞培养
HBMECs于含有10%FBS、10%Nu血清的RPMI-1640培养液中,于37℃、5%CO2、100%湿度的条件下培养。
(三)E44:△cglD基因缺失突变株的构建
将携带打靶基因两侧DNA片段的自杀性质粒pCVD442通过细菌间的接合传递到E44中,利用菌体内同源交换,从E44中剔除cglD基因。
(四)互补实验
将cglD基因的ORF克隆到质粒pFN476上,将其与pGP1-2共同转化给E44:△cglD。
(五)细菌黏附和侵袭试验
1、细菌黏附试验
向HBMECs单层加入1×107个过夜培养的大肠杆菌,作用1.5h后,收集细胞内外的细菌,结果用相对黏附力表示；
2、细菌侵袭试验
向HBMECs单层加入1×107个过夜培养的大肠杆菌,作用1.5h后,杀死细胞外的细菌,收集细胞内的细菌,结果用相对侵袭力表示。
(六)半数致死量和生存分析
Bliss法计算大肠杆菌对新生鼠的半数致死量；Kaplan-Meier方法绘制生存曲线。
(七)新生大鼠实验性细菌性脑膜炎模型的建立
皮下注射一定浓度的细菌,建立新生大鼠菌血症模型,脑膜炎模型由血行播散而致。
(八)脑脊液白细胞计数及蛋白和乳酸含量的测定
血细胞计数板计数脑脊液白细胞,BCA蛋白定量法测定脑脊液蛋白含量,乳酸脱氢酶比色法测定脑脊液乳酸含量。
(九)组织病理学分析
新生大鼠脑切片经HE染色后,显微镜观察脑膜以及大脑皮层区中性粒细胞的浸润。
(十)人多形核白细胞(PMNs)跨内皮迁移实验
从人新鲜的外周血中分离PMNs,计数经大肠杆菌趋化后穿过HBMECs单层的PMNs。
(十一)重组蛋白的表达和纯化
利用BL21(DE3)表达含有cglD基因的重组质粒,用亲和层析法纯化融合蛋白。
(十二)甘油脱氢酶活性分析
比色法测定菌体裂解液和纯化蛋白的甘油脱氢酶活性。
二、大肠杆菌毒力岛基因IbeT的功能研究
(一)细菌培养
大肠杆菌以LB培养基培养,在行细胞侵袭试验前,用含有利福平的BHI培养基培养E44。
(二)细胞培养
HBMECs于含有10%FBS、10%Nu血清的RPMI-1640培养液中,于37℃、5%CO2、100%湿度的条件下培养。
(三)E44:△ibeT基因缺失突变株的构建
将携带打靶基因两侧DNA片段的自杀性质粒pCVD442通过细菌间的接合传递到E44中,利用菌体内同源交换,从E44中剔除ibeT基因。
(四)互补实验
将ibeT基因的ORF克隆到质粒pFN476上,将其与pGP1-2共同转化给E44：△ibeT。
(五)细菌黏附和侵袭试验
1、细菌黏附试验
向HBMECs单层加入1×107个过夜培养的大肠杆菌,作用1.5h后,收集细胞内外的细菌,结果用相对黏附力表示；
2、细菌侵袭试验
向HBMECs单层加入1×107个过夜培养的大肠杆菌,作用1.5h后,杀死细胞外的细菌,收集细胞内的细菌,结果用相对侵袭力表示。
(六)细菌在细胞内存活试验
向HBMECs单层加入1×107个过夜培养的大肠杆菌,作用1.5h后,杀死细胞外的细菌,在继续培养指定时间收集细胞内的细菌,结果用菌落数表示。
(七)细菌生长曲线测定
将细菌接种于一定体积的培养基中培养,测定大肠杆菌在不同时间点波长为600 nm时的吸光度值,绘制生长曲线。
(八)免疫荧光实验
激光共聚焦扫描显微镜进行观察。
(九)透射电镜观察
利用超薄切片机将包埋块切成70～80nm的超薄切片,经染色后,利用透射电子显微镜观察。
(十)细菌缓冲容量测定
通过测定细菌胞内和胞外总的缓冲容量和细菌胞外缓冲容量,计算得到细菌胞内缓冲容量。
结果一、大肠杆菌毒力岛基因cgID在实验性新生儿脑膜炎中的作用
1、E.coli Kl等位基因cglD缺失突变株的构建
2、cglD基因缺失延长脑膜炎模型鼠的存活时间,但并不影响E.coliK1穿过血脑屏障
3、cglD基因的缺失减轻了脑膜炎模型鼠脑脊液的改变
4、cglD基因的缺失减轻了脑膜炎模型鼠脑膜和脑皮质区中性粒细胞的浸润
5、CglD蛋白具有甘油脱氢酶的活性
二、毒力岛基因ibeT有助于大肠杆菌抵抗人脑微血管内皮细胞溶酶体的降解
1、E.coli K1等位基因ibeT缺失突变株的构建
2、E.coli K1 ibeT基因缺失影响E.coli K1侵袭HBMECs
3、ibeT基因缺失抑制了E.coli K1在HBMECs中的生长
4、ibeT基因缺失导致E.coli K1逃逸HBMECs溶酶体的能力发生缺陷
5、ibeT基因有利于E.coli K1从ECV中逃逸入细胞浆
6、敲除ibeT基因后的E44:△ibeT突变株菌体胞内的缓冲容量下降
结论
毒力岛基因cglD作为E.coli K1的毒力因子,有利于E.coli K1的体内毒力,参与脑膜炎的炎症反应,但与穿越血脑屏障无关,cglD蛋白具有甘油脱氢酶的活性。
毒力岛侵袭基因ibeT有利于大肠杆菌降解ECV膜,避免与溶酶体融合,进而促使大肠杆菌逃逸进入细胞浆并进行复制。
Purpose
Bacterial 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.
Methods
1. Functional study of Escherichia coti K1 pathogenicity island gene cglD
(1) Bacterial culture
Bacterial strains were grown in LB medium; before bacterial invasion assay, E44 were grown in BHI medium.
(2) Cell culture
HBMECs 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 strain
Plasmid pCVD-△cglD was introduced into E44 by plate mating. E44:△cglD were constructed by bacterial homologous exchange.
(4) Complementation analysis
ORF of cglD gene was cloned into pFN476 vector and were transformed with pGP1-2 together into E44:△cglD.
(5) Bacterial adhesion and invasion assays
Bacterial 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 analysis
LD50 was determined by Bliss method and survival analysis were determined by Kaplan-Meier survival curve.
(7) Experimental rat model of neonatal meningitis
Bacteremia 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 CSF
WBC 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 assay
Fresh human PMNs were isolated from human peripheral blood. Migrated PMNs were counted in a hemacytometer.
(11) Recombinant protein expression and purification
Recombinant plasmid was expressed in BL21 (DE3) and GST-CglD was purified by affinity chromatography.
(12) Glycerol dehydrogenase (GLDH) assay
The activity of glycerol dehydrogenase was determined by colorimetric methods.
2. Functional study of Escherichia coli Kl pathogenicity island gene ibeT
(1) Bacterial culture
Bacterial strains were grown in LB medium, before bacterial invasion assay, E44 were grown in BHI medium.
(2) Cell culture
HBMECs were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 10% Nu serum.
(3) Construction of isogenic in-frame deletion mutant strain
Plasmid pCVD-△ibeT was introduced into E44 by plate mating. E44:△ibeT were constructed by bacterial homologous exchange.
(4) Complementation analysis
ORF of ibeT gene was cloned into pFN476 vector and were transformed with pGPl-2 together into E44:△ibeT.
(5) Bacterial adhesion and invasion assays
Bacterial 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 curves
Bacterial 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 bacteria
Cytoplasmic buffering capacity was calculated by the difference between total buffering capacity and extracellular buffering capacity.
Results
1. 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.
Conclusion
CglD 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.
新生儿细菌性脑膜炎是儿科严重感染性疾病之一,临床上尽管可以选用相应的抗生素治疗,但是病死率仍然得不到显著的改善。幸存者常伴有失听、失明、癫痫、智力和运动障碍后遗症。因此,探讨有效的新生儿细菌性脑膜炎的防治策略具有重要意义。
大肠杆菌(Escherichia coli, E. coli)是导致新生儿脑膜炎最常见的革兰氏阴性致病菌。从脑膜炎患儿脑脊液中分离的大肠杆菌中,80%以上均带有K1荚膜。现已清楚,E.coli K1通过血行播散入脑而引起脑膜炎。但是,血液中的E.coli K1如何穿过主要由脑微血管内皮细胞(brain microvascular endothelial cells, BMECs)组成的血脑屏障(blood brain barrier, BBB),迄今尚不清楚。借助于体外分离培养的人脑微血管内皮细胞(human brain microvascular endothelial cells, HBMECs)单层——血脑屏障模型,学者们先后鉴定了E. coli Kl中的参与侵袭HBMECs相关基因,如fimH, ompA, ibeA, ibeB, CNF1等,并对参与E.coli K1侵袭相关的细胞内信号转导通路如PI3K、PKB、PKC、RhoGTPase以及FAK等细胞骨架组装调控信号分子等进行了探讨。但已鉴定的这些E. coli K1穿过BBB的“决定因子”,特别是每个E. coli K1侵袭基因介导细菌侵袭HBMECs的功能差异、联系、及详细机制尚有待进一步研究。
我们在先前的研究中,从致新生儿脑膜炎E.coli K1株RS218(O18:K1:H7)中克隆鉴定了一个E.coli K1毒力岛基因GimA (genetic island of meningitic E.coli containing ibeA)(图1)。GimA全长20.3kb,仅存在于E.coli K1,其他非脑膜炎大肠杆菌中如E.coli K12均不含GimA序列。GimA含有四个操纵子,编码15个基因：(1)ptnIPKC (GimAl),包括ptnI、ptnP、ptnK、ptnC;(2)CglDTEC(GimA2),包括cglD、cglT、cglE、cglC;(3)GcxKRCI(GimA3),包括gcxK、gcxR、gcxC、gcxI; (4) IbeRAT(GimA4),包括ibeR、ibeA、ibeT。其中,ibeA基因的功能已得到初步研究,它在Kcoki K1侵袭HBMECs中起作用；其余14个基因的功能尚不清楚。GimA中推断的各基因编码蛋白与目前GeneBank库中功能已知蛋白之间的同源性高低程度分析结果提示：GimAl中ptnl与磷酸烯醇型丙酮酸磷酸化转移酶有很高的同源性；GimA2中cglD与甘油脱氢酶的同源性为62%;GimA3中gcxC与乙醛酸.醛连接酶的同源性高达67%;GimA4中含有E.coli K1侵袭HBMECs有关的ibeA,而其中的两个基因ibeT和ibeR尚未发现与其他蛋白有较高的同源性,鉴于这两个基因与ibeA同处于一个操纵子,故提示可能参与E.coli K1侵袭HBMECs.探讨这些基因在Ecoli Kl致脑膜炎中的作用,可以使我们更好的了解大肠杆菌性脑膜炎的发病机制。因此,我们选取了GimA2中的cglD基因和GimA4中的ibeT基因作为研究对象,展开了本工作。
方法
一、大肠杆菌毒力岛基因cglD的功能研究
(一)细菌培养
大肠杆菌以LB培养基培养,在行细胞侵袭试验前,用含有利福平的BHI培养基培养E44。
(二)细胞培养
HBMECs于含有10%FBS、10%Nu血清的RPMI-1640培养液中,于37℃、5%CO2、100%湿度的条件下培养。
(三)E44:△cglD基因缺失突变株的构建
将携带打靶基因两侧DNA片段的自杀性质粒pCVD442通过细菌间的接合传递到E44中,利用菌体内同源交换,从E44中剔除cglD基因。
(四)互补实验
将cglD基因的ORF克隆到质粒pFN476上,将其与pGP1-2共同转化给E44:△cglD。
(五)细菌黏附和侵袭试验
1、细菌黏附试验
向HBMECs单层加入1×107个过夜培养的大肠杆菌,作用1.5h后,收集细胞内外的细菌,结果用相对黏附力表示；
2、细菌侵袭试验
向HBMECs单层加入1×107个过夜培养的大肠杆菌,作用1.5h后,杀死细胞外的细菌,收集细胞内的细菌,结果用相对侵袭力表示。
(六)半数致死量和生存分析
Bliss法计算大肠杆菌对新生鼠的半数致死量；Kaplan-Meier方法绘制生存曲线。
(七)新生大鼠实验性细菌性脑膜炎模型的建立
皮下注射一定浓度的细菌,建立新生大鼠菌血症模型,脑膜炎模型由血行播散而致。
(八)脑脊液白细胞计数及蛋白和乳酸含量的测定
血细胞计数板计数脑脊液白细胞,BCA蛋白定量法测定脑脊液蛋白含量,乳酸脱氢酶比色法测定脑脊液乳酸含量。
(九)组织病理学分析
新生大鼠脑切片经HE染色后,显微镜观察脑膜以及大脑皮层区中性粒细胞的浸润。
(十)人多形核白细胞(PMNs)跨内皮迁移实验
从人新鲜的外周血中分离PMNs,计数经大肠杆菌趋化后穿过HBMECs单层的PMNs。
(十一)重组蛋白的表达和纯化
利用BL21(DE3)表达含有cglD基因的重组质粒,用亲和层析法纯化融合蛋白。
(十二)甘油脱氢酶活性分析
比色法测定菌体裂解液和纯化蛋白的甘油脱氢酶活性。
二、大肠杆菌毒力岛基因IbeT的功能研究
(一)细菌培养
大肠杆菌以LB培养基培养,在行细胞侵袭试验前,用含有利福平的BHI培养基培养E44。
(二)细胞培养
HBMECs于含有10%FBS、10%Nu血清的RPMI-1640培养液中,于37℃、5%CO2、100%湿度的条件下培养。
(三)E44:△ibeT基因缺失突变株的构建
将携带打靶基因两侧DNA片段的自杀性质粒pCVD442通过细菌间的接合传递到E44中,利用菌体内同源交换,从E44中剔除ibeT基因。
(四)互补实验
将ibeT基因的ORF克隆到质粒pFN476上,将其与pGP1-2共同转化给E44：△ibeT。
(五)细菌黏附和侵袭试验
1、细菌黏附试验
向HBMECs单层加入1×107个过夜培养的大肠杆菌,作用1.5h后,收集细胞内外的细菌,结果用相对黏附力表示；
2、细菌侵袭试验
向HBMECs单层加入1×107个过夜培养的大肠杆菌,作用1.5h后,杀死细胞外的细菌,收集细胞内的细菌,结果用相对侵袭力表示。
(六)细菌在细胞内存活试验
向HBMECs单层加入1×107个过夜培养的大肠杆菌,作用1.5h后,杀死细胞外的细菌,在继续培养指定时间收集细胞内的细菌,结果用菌落数表示。
(七)细菌生长曲线测定
将细菌接种于一定体积的培养基中培养,测定大肠杆菌在不同时间点波长为600 nm时的吸光度值,绘制生长曲线。
(八)免疫荧光实验
激光共聚焦扫描显微镜进行观察。
(九)透射电镜观察
利用超薄切片机将包埋块切成70～80nm的超薄切片,经染色后,利用透射电子显微镜观察。
(十)细菌缓冲容量测定
通过测定细菌胞内和胞外总的缓冲容量和细菌胞外缓冲容量,计算得到细菌胞内缓冲容量。
结果一、大肠杆菌毒力岛基因cgID在实验性新生儿脑膜炎中的作用
1、E.coli Kl等位基因cglD缺失突变株的构建
2、cglD基因缺失延长脑膜炎模型鼠的存活时间,但并不影响E.coliK1穿过血脑屏障
3、cglD基因的缺失减轻了脑膜炎模型鼠脑脊液的改变
4、cglD基因的缺失减轻了脑膜炎模型鼠脑膜和脑皮质区中性粒细胞的浸润
5、CglD蛋白具有甘油脱氢酶的活性
二、毒力岛基因ibeT有助于大肠杆菌抵抗人脑微血管内皮细胞溶酶体的降解
1、E.coli K1等位基因ibeT缺失突变株的构建
2、E.coli K1 ibeT基因缺失影响E.coli K1侵袭HBMECs
3、ibeT基因缺失抑制了E.coli K1在HBMECs中的生长
4、ibeT基因缺失导致E.coli K1逃逸HBMECs溶酶体的能力发生缺陷
5、ibeT基因有利于E.coli K1从ECV中逃逸入细胞浆
6、敲除ibeT基因后的E44:△ibeT突变株菌体胞内的缓冲容量下降
结论
毒力岛基因cglD作为E.coli K1的毒力因子,有利于E.coli K1的体内毒力,参与脑膜炎的炎症反应,但与穿越血脑屏障无关,cglD蛋白具有甘油脱氢酶的活性。
毒力岛侵袭基因ibeT有利于大肠杆菌降解ECV膜,避免与溶酶体融合,进而促使大肠杆菌逃逸进入细胞浆并进行复制。
Purpose
Bacterial 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.
Methods
1. Functional study of Escherichia coti K1 pathogenicity island gene cglD
(1) Bacterial culture
Bacterial strains were grown in LB medium; before bacterial invasion assay, E44 were grown in BHI medium.
(2) Cell culture
HBMECs 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 strain
Plasmid pCVD-△cglD was introduced into E44 by plate mating. E44:△cglD were constructed by bacterial homologous exchange.
(4) Complementation analysis
ORF of cglD gene was cloned into pFN476 vector and were transformed with pGP1-2 together into E44:△cglD.
(5) Bacterial adhesion and invasion assays
Bacterial 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 analysis
LD50 was determined by Bliss method and survival analysis were determined by Kaplan-Meier survival curve.
(7) Experimental rat model of neonatal meningitis
Bacteremia 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 CSF
WBC 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 assay
Fresh human PMNs were isolated from human peripheral blood. Migrated PMNs were counted in a hemacytometer.
(11) Recombinant protein expression and purification
Recombinant plasmid was expressed in BL21 (DE3) and GST-CglD was purified by affinity chromatography.
(12) Glycerol dehydrogenase (GLDH) assay
The activity of glycerol dehydrogenase was determined by colorimetric methods.
2. Functional study of Escherichia coli Kl pathogenicity island gene ibeT
(1) Bacterial culture
Bacterial strains were grown in LB medium, before bacterial invasion assay, E44 were grown in BHI medium.
(2) Cell culture
HBMECs were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 10% Nu serum.
(3) Construction of isogenic in-frame deletion mutant strain
Plasmid pCVD-△ibeT was introduced into E44 by plate mating. E44:△ibeT were constructed by bacterial homologous exchange.
(4) Complementation analysis
ORF of ibeT gene was cloned into pFN476 vector and were transformed with pGPl-2 together into E44:△ibeT.
(5) Bacterial adhesion and invasion assays
Bacterial 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 curves
Bacterial 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 bacteria
Cytoplasmic buffering capacity was calculated by the difference between total buffering capacity and extracellular buffering capacity.
Results
1. 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.
Conclusion
CglD 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.
引文
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10 Sheng-He Huang, Ze-Sheng Wan, Yu-Hua Chen, et al. Further Characterization of Escherichia coli Brain Microvascular Endothelial Cell Invasion Gene ibeA by Deletion, Complementation, and Protein Expression The Journal of Infectious Diseases 2001; 183:1071-8
11 Germon P, Chen YH, He L,et al. ibeA, a virulence factor of avian pathogenic Escherichia coli. Microbiology.2006 Apr;151(Pt 4):1179-86.
12 SHENG-HE HUANG, YU-HUA CHEN, QI FU, et al. Identification and Characterization of an Escherichia coli Invasion Gene Locus, ibeB, Required for Penetration of Brain Microvascular Endothelial Cells INFECTION AND IMMUNITY, May 1999, p.2103-2109
13赵伟东,陈誉华,黄胜和.大肠杆菌脑微血.管内皮细胞侵袭基因ibeB的编码产物为外膜蛋白前体.中国生物化学与分子生物学报.2004年8月20(4)：497—501
14 YING WANG, SHENG-HE HUANG, CAROL A. WASS, et al. Application of Signature-Tagged Mutagenesis for Identification of Escherichia coli K1 Genes That Contribute to Invasion of Human Brain Microvascular Endothelial Cells INFECTION AND IMMUNITY, Sept.2000, p.5056-5061
15王淳,方文刚,陈誉华.大肠杆菌脑微血管内皮细胞侵袭蛋白-yijP的表达纯化.中国医科大学学报0258-4646(2005)
16 JILL A. HOFFMAN, JULIE L. BADGER, YAN ZHANG, et al. Escherichia coli K1 aslA Contributes to Invasion of Brain Microvascular Endothelial Cells In Vitro and In Vivo. INFECTION AND IMMUNITY, Sept.2000, p.5062-5067
17 Maurice J. Bessman, Joseph D. Walsh, Christopher A. Dunn, et al. The Gene ygdP, Associated with the Invasiveness of Escherichia coliK1, Designates a Nudix. Hydrolase, Orfl76, Active on Adenosine (5_)-Pentaphospho-(5_)-adenosine (Ap5A).THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol.276, No.41, Issue of October 12, pp.37834-37838,2001
18 [ill A. Hoffman, Julie L. Badger, Yan Zhang, et al. Escherichia coli K1 purA and sorC are preferentially expressed upon association with human brain microvascular endothelial cells Microbial Pathogenesis 2001; 31:69-79
19 Kim KJ, Chung JW, Kim KS.67-kDa laminin receptor promotes internalization of cytotoxic necrotizing factor 1-expressing Escherichia coli K1 into human brain microvascular endothelial cells. J Biol Chem.2006 Jan 14; 280(2):1360-8.
20 MARPADGA A. REDDY, CAROL A. WASS, KWANG SIK KIM, et al. Involvement of Focal Adhesion Kinase in Escherichia coli Invasion of Human Brain Microvascular
Endothelial Cells。INFECTION AND IMMUNITY, Nov.2000, p.6423-6430
21 Marpadga A. Reddy, Nemani V. Prasadarao, Carol A. Wass, et al. Phosphatidylinositol 3-Kinase Activation and Interaction with Focal Adhesion Kinase in Escherichia coli K1 Invasion of Human Brain Microvascular Endothelial Cells。THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol.275, No.47, Issue of November 24, pp.36769-36774, 2000
22 Sunil K. Sukumaran, George McNamara. Escherichia coli K-1 Interaction with Human Brain Micro-vascular Endothelial Cells Triggers Phospholipase C-_1 Activation Downstream of Phosphatidylinositol 3-Kinaseo THE JOURNAL OF BIOLOGICAL CHEMISTRY。Vol.278, No.46, Issue of November 14, pp.45753-45762,2003
23 Sunil K. Sukumaran, Nemani V. Prasadarao. Regulation of Protein Kinase C in Escherichia coli K1 Invasion of Human Brain Microvascular Endothelial Cells。THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol.277, No.14, Issue of April 5, pp.12253-12262,2002
24 Aninda Das,Liana Asatryan, Marpadga A. Reddy, et al. Differential Role of Cytosolic Phospholipase A2 in the Invasion of Brain Microvascular Endothelial Cells by Escherichia coli and Listeria monocytogenes。The Journal of Infectious Diseases 2001; 184:732-7
25 Rajyalakshmi S. Rudrabhatla, Sunil K. Sukumaran, Gary M. Bokoch, et al. Modulation of Myosin Light-Chain Phosphorylation by p21-Activated Kinase 1 in Escherichia coli Invasion of Human Brain Microvascular Endothelial Cells INFECTION AND IMMUNITY, May 2003, p.2787-2797
26 Rajyalakshmi S. Rudrabhatla, Suresh K. Selvaraj, NemaniV. Prasadarao. Role of Racl in Escherichia coli K1 invasion of human brain microvascular endothelial cells Microbes and Infection(2005)
27 Brian Y. Kim, Jay Kang, Kwang Sik Kim. Invasion processes of pathogenic Escherichia coli International Journal of Medical Microbiology 295 (2005) 463-470
28 Guang Wen Zhang,Naveed Ahmed Khan, Kee Jun Kim. Transforming growth factor-(3 increases Escherichia coli K1 adherence, invasion, and transcytosis in human brain microvascular endothelial cells. Cell Tissue Res (2002) 309:281-286
29 Yu-Hua Chen, Steven Han-Min Chen, Ambrose Jong, et al. Enhanced Escherichia coli invasion of human brain microvascular endothelial cells is associated with alternations in cytoskeleton induced by nicotine. Cellular Microbiology (2002) 4 (8),503-514
30 Kee Jun Kim, Simon Justin Elliott, Francescopaolo Di Cello, et al. The K1 capsule modulates trafficking of E. coli-containing vacuoles and enhances intracellular bacterial survival in human brain microvascular endothelial cells. Cellular Microbiology Volume 5 Issue 4 Page 245, April 2003
31 Zou Y, He L, Chi F, et al. Involvement of Escherichia coli K1 ibeT in bacterial adhesion that is associated with the entry into human brain microvascular endothelial cells. Med Microbiol Immunol,2008,197(4):337-344
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1黄邵良,陈述枚,何政贤.小儿内科学.北京：人民卫生出版社,2004.193-194
2 Huang S L, Chen S M, He Z X. Pediatric Internal Medicine. Beijing:People Health Publishing House,2004.193-194
3 Xie Y, Kim K J, Kim K S. Current concepts on Escherichia coli Kl translocation of the blood-brain barrier. FEMS Immunol Med Microbiol,2004,42(3):271-279
4 Huang S H, Stins M F, Kim K S. Bacterial penetration across the blood-brain barrier during the development of neonatal meningitis. Microbes Infect,2000,2(10):1237-1244
5 Huang S H, Chen Y H, Kong G, et al. A novel genetic island of meningitic Escherichia coli Kl containing the ibeA invasion gene (GimA):functional annotation and carbon-source-regulated invasion of human brain microvascular endothelial cells. Funct Integr Genomics,2001,1(5): 312-322
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7 Silver R P, Aaronson W, Sutton A, et al. Comparative analysis of plasmids and some metabolic characteristics of Escherichia coli K1 from diseased and healthy individuals. Infect Immun, 1980,29(1):200-206
8 Huang S H, Wan Z S, Chen Y H, et al. Further characterization of Escherichia coli brain microvascular endothelial cell invasion gene ibeA by deletion, complementation, and protein expression. J Infect Dis,2001,183(7):1071-1078
9 Huang S H, Chen Y H, Fu Q, et al. Identification and characterization of an Escherichia coli invasion gene locus, ibeB, required for penetration of brain microvascular endothelial cells. Infect Immun,1999,67(5):2103-2109
10 Santic M, Molmeret M, Barker J R, et al. A Francisella tularensis pathogenicity island protein essential for bacterial proliferation within the host cell cytosol. Cell Microbiol,2007,9(10): 2391-2403
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17 Santic M, Molmeret M, Klose K E, et al. Francisella tularensis travels a novel, twisted road within macrophages. Trends Microbiol,2006,14(1):37-44
18 Hackstadt T. Redirection of host vesicle trafficking pathways by intracellular parasites. Traffic, 2000,1(2):93-99
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1姚泰,主编生理学(第五版)。人民卫生出版社
2 Sheng-He Huang, Kwang Sik Kim. Bacterial penetration across the blood-brain barrier during the development of neonatal meningitis. Microbes and Infection,2,2000,1237-1244
3 Monique F. Stins, Julie Badger, Kwang Sik Kim. Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells Microbial Pathogenesis 2001; 30:19-28
4 Yi Xie, Kee Jun Kim, Kwang Sik Kim. Current concepts on Escherichia coli K1 translocation of the blood-brain barrier. FEMS Immunology and Medical Microbiology 42 (2004) 271-279
5 Nemani V. Prasadarao. Identification of Escherichia coli Outer Membrane Protein A Receptor on Human Brain Microvascular Endothelial Cells. INFECTION AND IMMUNITY, Aug. 2002,p.4556-4563
6 Sooan Shin, Gengshi Lu, Mian Cai., et al.Escherichia coli outer membrane protein A adheres to human brain microvascular endothelial cells Biochemical and Biophysical Research Communications 330 (2005) 1199-1204
7 Ying Wang, Zhang Guang Wen, Kwang Sik Kim. Role of S fimbriae in Escherichia coli K1 binding to brain microvascular endothelial cells in vitro and penetration into the central nervous system in vivo. Microbial Pathogenesis 37 (2004) 287-293
8 Teng CH, Cai M. Escherichia coli K1 RS218 interacts with human brain microvascular endothelial cells via type 1 fimbria bacteria in the fimbriated state. Infect Immun.2005 May;73(5):2923-31.
9 YING WANG, SHENG-HE HUANG, CAROL A. WASS, et al. The Gene Locus yijP Contributes to Escherichia coli K1 Invasion of Brain Microvascular Endothelial Cells INFECTION AND IMMUNITY, Sept.1999, p.4751-4756
10 Sheng-He Huang, Ze-Sheng Wan, Yu-Hua Chen, et al. Further Characterization of Escherichia coli Brain Microvascular Endothelial Cell Invasion Gene ibeA by Deletion, Complementation, and Protein Expression The Journal of Infectious Diseases 2001; 183:1071-8
11 Germon P, Chen YH, He L,et al. ibeA, a virulence factor of avian pathogenic Escherichia coli. Microbiology.2006 Apr;151(Pt 4):1179-86.
12 SHENG-HE HUANG, YU-HUA CHEN, QI FU, et al. Identification and Characterization of an Escherichia coli Invasion Gene Locus, ibeB, Required for Penetration of Brain Microvascular Endothelial Cells INFECTION AND IMMUNITY, May 1999, p.2103-2109
13赵伟东,陈誉华,黄胜和.大肠杆菌脑微血.管内皮细胞侵袭基因ibeB的编码产物为外膜蛋白前体.中国生物化学与分子生物学报.2004年8月20(4)：497—501
14 YING WANG, SHENG-HE HUANG, CAROL A. WASS, et al. Application of Signature-Tagged Mutagenesis for Identification of Escherichia coli K1 Genes That Contribute to Invasion of Human Brain Microvascular Endothelial Cells INFECTION AND IMMUNITY, Sept.2000, p.5056-5061
15王淳,方文刚,陈誉华.大肠杆菌脑微血管内皮细胞侵袭蛋白-yijP的表达纯化.中国医科大学学报0258-4646(2005)
16 JILL A. HOFFMAN, JULIE L. BADGER, YAN ZHANG, et al. Escherichia coli K1 aslA Contributes to Invasion of Brain Microvascular Endothelial Cells In Vitro and In Vivo. INFECTION AND IMMUNITY, Sept.2000, p.5062-5067
17 Maurice J. Bessman, Joseph D. Walsh, Christopher A. Dunn, et al. The Gene ygdP, Associated with the Invasiveness of Escherichia coliK1, Designates a Nudix. Hydrolase, Orfl76, Active on Adenosine (5_)-Pentaphospho-(5_)-adenosine (Ap5A).THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol.276, No.41, Issue of October 12, pp.37834-37838,2001
18 [ill A. Hoffman, Julie L. Badger, Yan Zhang, et al. Escherichia coli K1 purA and sorC are preferentially expressed upon association with human brain microvascular endothelial cells Microbial Pathogenesis 2001; 31:69-79
19 Kim KJ, Chung JW, Kim KS.67-kDa laminin receptor promotes internalization of cytotoxic necrotizing factor 1-expressing Escherichia coli K1 into human brain microvascular endothelial cells. J Biol Chem.2006 Jan 14; 280(2):1360-8.
20 MARPADGA A. REDDY, CAROL A. WASS, KWANG SIK KIM, et al. Involvement of Focal Adhesion Kinase in Escherichia coli Invasion of Human Brain Microvascular
Endothelial Cells。INFECTION AND IMMUNITY, Nov.2000, p.6423-6430
21 Marpadga A. Reddy, Nemani V. Prasadarao, Carol A. Wass, et al. Phosphatidylinositol 3-Kinase Activation and Interaction with Focal Adhesion Kinase in Escherichia coli K1 Invasion of Human Brain Microvascular Endothelial Cells。THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol.275, No.47, Issue of November 24, pp.36769-36774, 2000
22 Sunil K. Sukumaran, George McNamara. Escherichia coli K-1 Interaction with Human Brain Micro-vascular Endothelial Cells Triggers Phospholipase C-_1 Activation Downstream of Phosphatidylinositol 3-Kinaseo THE JOURNAL OF BIOLOGICAL CHEMISTRY。Vol.278, No.46, Issue of November 14, pp.45753-45762,2003
23 Sunil K. Sukumaran, Nemani V. Prasadarao. Regulation of Protein Kinase C in Escherichia coli K1 Invasion of Human Brain Microvascular Endothelial Cells。THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol.277, No.14, Issue of April 5, pp.12253-12262,2002
24 Aninda Das,Liana Asatryan, Marpadga A. Reddy, et al. Differential Role of Cytosolic Phospholipase A2 in the Invasion of Brain Microvascular Endothelial Cells by Escherichia coli and Listeria monocytogenes。The Journal of Infectious Diseases 2001; 184:732-7
25 Rajyalakshmi S. Rudrabhatla, Sunil K. Sukumaran, Gary M. Bokoch, et al. Modulation of Myosin Light-Chain Phosphorylation by p21-Activated Kinase 1 in Escherichia coli Invasion of Human Brain Microvascular Endothelial Cells INFECTION AND IMMUNITY, May 2003, p.2787-2797
26 Rajyalakshmi S. Rudrabhatla, Suresh K. Selvaraj, NemaniV. Prasadarao. Role of Racl in Escherichia coli K1 invasion of human brain microvascular endothelial cells Microbes and Infection(2005)
27 Brian Y. Kim, Jay Kang, Kwang Sik Kim. Invasion processes of pathogenic Escherichia coli International Journal of Medical Microbiology 295 (2005) 463-470
28 Guang Wen Zhang,Naveed Ahmed Khan, Kee Jun Kim. Transforming growth factor-(3 increases Escherichia coli K1 adherence, invasion, and transcytosis in human brain microvascular endothelial cells. Cell Tissue Res (2002) 309:281-286
29 Yu-Hua Chen, Steven Han-Min Chen, Ambrose Jong, et al. Enhanced Escherichia coli invasion of human brain microvascular endothelial cells is associated with alternations in cytoskeleton induced by nicotine. Cellular Microbiology (2002) 4 (8),503-514
30 Kee Jun Kim, Simon Justin Elliott, Francescopaolo Di Cello, et al. The K1 capsule modulates trafficking of E. coli-containing vacuoles and enhances intracellular bacterial survival in human brain microvascular endothelial cells. Cellular Microbiology Volume 5 Issue 4 Page 245, April 2003
31 Zou Y, He L, Chi F, et al. Involvement of Escherichia coli K1 ibeT in bacterial adhesion that is associated with the entry into human brain microvascular endothelial cells. Med Microbiol Immunol,2008,197(4):337-344