The Identification Of The Signal Pathway Involved In Enterobacter Sakazakii Invasion Of Human Brain Microvascular Endothelial Cells

Objective:Enterobacter sakazakii(E.sakazakii)is an opportunistic pathogen that causes an often fatal form of meningitis,meningoencephalitis,necrotizing enterocolitis,and sepsis in neonates and infants,with a case fatality rate of 40%-80%.Besides the high rate of mortality,the brain infections due to E.sakazakii often lead to permanent impairments in mental and physical capabilities in surviving patients.Contaminated powdered infant formula repr-esents the only known source of infection in neonates.A recent outbreak of neonatal infection caused by E.sakazakii and the recall of E.sakazakii-contaminated infant formula preparations in the Unites States have stimulated a renewed research interest on this pathogen.The blood-brain barrier(BBB)is mainly composed of brain microvascular endothelial cell,neuroglia membrane and inner basement membrane.The BBB is a metabolic and physiological barrier between the brain and the circulatory system,which main function of the blood brain barrier to prevent toxic substances in the circulation such as drugs,toxins into the brain,the brain to maintain relatively constant internal environment thus,the protection of the central nervous system damage.The human brain microvascular endothelial cells(HBMECs)were isolated and cultured in vitro to simulate the blood brain barrier,which can be used as an ideal object to study the function of blood brain barrier.Some bacteria can secrete effector molecules in the host cells nearby,caused by changes of cytoskeleton in host cells,conducive to pathogen changes through the host cell cytoskeleton and adhesion to the host cell surface,invasion into host cells and in host cells and host cell migration,and finally the formation of pinocytotic vesicles,avoid swallowed.Several pathogenic bacteria that cause central nervous system such as Escherichia coli K1,Citrobacter freundii,Listeria monocytogenes and Group B streptococcus invade human brain microvascular endothelial cells(HBMECs),an in vitro model of the blood–brain barrier.To assess the role of E.sakazakii in human disease,evaluation of potential virulence factors is required.However,at this time very little is known about the of microbial pathogens to express adherence factors responsible for recognizing and binding to specific receptor moieties of cell,thereby enabling the bacteria to resist host strategies that would impede colonization.Kim et al demonstrated that activation of protein kinase C-a,PI3-kinase,and caveolin-1 is required for the condensation of actin filaments during E.coli invasion HBMECs.However,mechanisms involved in the entry of E.sakazakii into HBMECs are not clearly known.In an effort to understand the pathogenesis of meningitis caused by E.sakazakii.How does E.sakazakii invasion HBMECs? Does actin-filment take part in mechanisms involved in the entry of E.sakazakii into HBMECs? What role does the upregulation of PI3 K and ERK in HBMECs play in E.sakazakii invasion HBMECs? These problems and the pathogenic mechanism of Enterobacter sakazakii is closely related with the solution of the above problems,we can find the methods of prevention and treatment of Enterobacter sakazakii meningitis caused by other related diseases of the nervous system,as a solid theoretical basis for further study on the pathogenic mechanism of Enterobacter sakazakii.So I did some relevant experiments to resolve above questions.Methods: 1.Study on changes of cytoskeleton induced by kazakhazakin and its effect on HBMECs and its effect on HBMECs1.1 Cell culture: HBMECs is cultured in RPMI 1640 culture medium containing 10% newborn bovine serum and fetal bovine serum etc.1.2 Bacterial culture:ES 29544 was obtained from ATCC and was grown in LB or Tryptic Soy Broth medium without any antibiotics.1.3 Invasion Assays.E.sakazakii ?E44?HB101 binding and invasion assays using HBMECs.1.4 Invasion Assays.HBMECs were pretreated with inhibitor cytoD—filment inhibitor for 30 min prior to the addition of bacteria.1.5 Invasion Assays.HBMECs were pretreated with inhibitor colchine—tubulin inhibitor for 30 min prior to the addition of bacteria.1.6 Immunofluorescence Staining.Filmentous actin is detected by rhodamin-phallodin when E.sakazakii invasion HBMECs.1.7 Immunofluorescence Staining.The ?-tubulin is detected when E.sakazakii invasion HBMECs.2.In study of PI3 K signaling pathways in human brain microvascular endothelial cells for E.sakazakii invasion.2.1 Cell culture: HBMECs is cultured in RPMI 1640 culture medium containing 10% newborn bovine serum and fetal bovine serum etc.2.2 Bacterial culture:ES 29544 was obtained from ATCC and was grown in LB or Tryptic Soy Broth medium without any antibiotics.2.3 Invasion Assays.2.3.1 HBMECs were pretreated with inhibitor LY294002 and Wortmanin-PI3 K inhibitor for 30 min prior to the addition of bacteria.2.3.2 HBMECs were pretreated with inhibitor G?6976—PKC inhibitor for 30 min prior to the addition of bacteria.2.3.3 HBMECs were pretreated with inhibitor PP1 and PP2—Src inhibitor for 30 min prior to the addition of bacteria.2.3.4 HBMECs were pretreated with inhibitor Y27632—ROCK inhibitor for 30 min prior to the addition of bacteria.2.4 Western-blot:2.4.1 The phosphorelation level of AKT/PKB is detected by western-blot when E.sakazakii invasion HBMECs in different time.2.4.2 The phosphorelation level of AKT/PKB is detected by western-blot when HBMECs were pretreated with inhibitor LY294002 and Wortmanin-PI3 K inhibitor for 30 min prior to the addition of bacteria.2.5 Immunofluorescence Staining.Filmentous actin is detected by rhodamin-phallodin when HBMECs were pretreated with inhibitor LY294002 and Wortmanin-PI3 K inhibitor for 30 min prior to the addition of bacteria.2.6 Immunofluorescence Staining.The ?-tubulin is detected when HBMECs were pretreated with inhibitor LY294002 and wortmanin-PI3 K inhibitor for 30 min prior to the addition of bacteria.2.7 Dominant-negative PI3K(?p110)can inhibited the invasion of Enterobacter sakazakii to HBMECs and eliminate the change of F-actin and akt phosphorylation caused by E.sakazakii invasion.2.7.1 Invasion assay was used to exam the bacterial invasion ability to the dominant-negative PI3 K HBMECs.2.7.2 Western-blot methods were used to exam the PI3 K expression on dominant-negative PI3 K HBMECs.2.7.3 Immunofluorescence methods were used to detect the change of the cytoskeletal microfilament.2.8 Invasion Assays,Immunofluorescence Staining,Western-blot methods were used to exam cPLA2 a association with HBMECs for E.sakazakii invasion 3 In study of ERK signaling pathways in human brainmicrovascular endothelial cells for E.sakazakii invasion3.1 Cell culture: HBMECs is cultured in RPMI 1640 culture medium containing 10% newborn bovine serum and fetal bovine serum etc.3.2 Bacterial culture:ES 29544 was obtained from ATCC and was grown in LB or Tryptic Soy Broth medium without any antibiotics.3.3 Invasion Assays.HBMECs were pretreated with inhibitor PD98059-ERKinhibitor for 30 min prior to the addition of bacteria.3.4 Western-blot:3.4.1 The phosphorelation level of ERK is detected by western-blot when Enterobacter sakazakii invasion HBMECs in different time.3.4.2 The phosphorelation level of ERK is detected by western-blot when HBMECs were pretreated with inhibitor PD98059-ERK inhibitor for 30 min prior to the addition of bacteria.3.5 Immunofluorescence Staining.Filmentous actin is detected by rhodamin-phallodin when HBMECs were pretreated with inhibitor PD98059-ERK inhibitor for 30 min prior to the addition of bacteria.3.6 Immunofluorescence Staining.The ?-tubulin is detected when HBMECs were pretreated with inhibitor PD98059-ERK inhibitor for 30 min prior to the addition of bacteria.3.7 ERK siRNAs.3.7.1 ERK siRNAs were transiently transfected into HBMECs.3.7.2 ERK expressions are detected by western-blot in the above transfected HBMECs.3.7.3 Filmentous actin is detected by rhodamin-phallodin through the above transfected HBMECs monolayer.Results:1 In study of induce microfilment and tubulin condensation in human brainmicrovascular endothelial cells for E.sakazakii invasion.1.1 The biological characteristics of E.sakazakii.E.sakazakii.colonies exhibited a blue-green color during culture on DFI(a specific commercial chromogenic agar medium)agar plates.1.2 E.sakazakii can invasive HBMECs.The E.sakazakii have a similar invasion ability as an identified meningitis E.coli K1 to HBMECs.1.3 Cytochalasin D,an inhibitor of microfilament function,can inhibited the invasion of E.sakazakii to HBMECs without affect on bacterial adherence.Cytochalasin D has dose dependent inhibition on the invasion of E.sakazakii to HBMECs.1.4 Colchicine,an inhibitor of microtubule function,can inhibited the invasion of E.sakazakii to HBMECs without affect on bacterial adherence.Colchicine has dose dependent inhibition on the invasion of E.sakazakii to HBMECs.1.5 E.sakazakii may lead to F-actin cytoskeletal change in HBMECs.Invasion of E.sakazakii to HBMECs induced microfilament depolymerization and redistribution of HBMECs1.6 E.sakazakii may lead to microtubule cytoskeletal change in HBMECs.Invasion of E.sakazakii to HBMECs induced microtubule depolymerization and redistribution of HBMECs.2.Invasion of E.sakazakii to HBMECs is associated with the activation of the PI3 K pathways.2.1 PI3 K inhibitor LY294002 and Wortmannin strongly suppressed the invasion of E.sakazakii to HBMECs,PKC inhibitor G?6976 inhibited the invasion of E.sakazakii to HBMECs;The inhibitor of Src and Rock can not block the invasion of E.sakazakii to HBMECs.2.1.1 PI3 K inhibitor LY294002 and Wortmannin suppressed the invasion of E.sakazakii to HBMECs in a dose-dependent manner.2.1.2 PKC inhibitor G?6976 suppressed the invasion of E.sakazakii to HBMECs in a dose-dependent manner.2.1.3 Src inhibitor PP1 and PP2 do not affect on the invasion of E.sakazakii to HBMECs.2.1.4 ROCK inhibitor Y27632 do not affect on the invasion of E.sakazakii to HBMECs2.2 Invasion of E.sakazakii to HBMECs lead to the activation of the PI3 K pathways.2.2.1 Invasion of E.sakazakii to HBMECs for indicated time induced Akt phosphorylation.Akt phosphorylation on HBMECs was obvious elevated at 15min detected by Western blot.2.2.2 Confluent HBMECs were pretreated with LY294002 or Wortmannin for 30 min,and western blot detected no change of the p-Akt expression.2.3 PI3 K inhibitor eliminates the change of microfilament in HBMECs caused by the invasion of E.sakazakii.2.4 PI3 K inhibitor eliminates the change of microtubule in HBMECs caused by the invasion of E.sakazakii.2.5 Dominant-negative PI3K(?p110)can inhibited the invasion of E.sakazakii to HBMECs and eliminate the change of F-actin and Akt phosphorylation caused by E.sakazakii invasion.2.5.1 Invasion assay was used to exam the bacterial invasion ability to the dominant-negative PI3 K HBMECs.The result showed that dominant-negative PI3K(?p110)can inhibited the invasion of E.sakazakii to HBMECs.2.5.2 Western-blot methods were used to exam the PI3 K expression on dominant-negative PI3 K HBMECs.The results showed that dominant-negative PI3K(?p110)can eliminate the akt phosphorylation caused by E.sakazakii invasion.2.5.3 Immunofluorescence methods were used to detect the change of the cytoskeletal microfilament.The results showed that dominant-negative PI3K (?p110)can eliminate the change of F-actin caused by E.sakazakii invasion by immunofluorescence.2.6 cPLA2 a is associated with HBMECs for E.sakazakii invasion.2.7 cPLA2 a is akt for E.sakazakii invade HBMECs downstream effectors.3.In study of ERK signaling pathways in human brain microvascular endothelial cells for E.sakazakii invasion.3.1 ERK inhibitor PD98059 inhibited the invasion of E.sakazakii to HBMECs ERK inhibitor PD98059 suppressed the invasion of E.sakazakii to HBMECs in a dose-dependent manner.3.2 Invasion of Enterobacter sakazakii to HBMECs lead to the activation of the ERK pathways.3.2.1 Invasion of E.sakazakii to HBMECs for indicated time induced ERK phosphorylation.ERK phosphorylation on HBMECs was obvious elevated at 10min detected by Western blot.3.2.2 Confluent HBMECs were pretreated with PD98059 for 30 min,and western blot detected no change of the ERK phosphorylation expression.3.3 ERK inhibitor eliminates the change of microfilament in HBMECs caused by the invasion of E.sakazakii.3.4 ERK inhibitor eliminates the change of microtubule in HBMECs caused by the invasion of E.sakazakii.3.5 Transient transected HBMECs with ERK siRNAs may inhibit the invasion ability of E.sakazakii and further eliminate the change of microtubule in HBMECs caused by the invasion of the bacteria.CONCLUSIONS: E.sakazakii can invasion HBMECs.Actin-filment and ?-tubulin take part in mechanisms involved in the entry of E.sakazakii into HBMECs,and through activates PI3K/AKT and MAPK signaling molecules...