Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells

1
Bacterial invasion and transcytosis in
transfected human brain microvascular endothelial
cells

• Stins MF, Badger J, Kim SK. 2001. Microbial
  Pathogenesis. 3019-28.
• Presented by Jess Jung

2
General Rationale

• High fatality rate of meningitis
• Incomplete knowledge of pathogenesis
• Lack of reliable blood-brain barrier (BBB) model

3
Purpose

• To develop an immortalized human brain
  microvascular endothelial cell (HBMEC) line to
  further examine and understand the pathogenesis
  of bacteria-causing meningitis (Escherichia coli
  and Citrobacter freundii)

4
Review of Terms

• Escherichia coli (E. coli)
• gram-negative bacilli
• Enterobacteriaceae
• Causative agent of many bacterial infections
• Citrobacter freundii (C. freundii)
• gram-negative bacilli
• Enterobacteriaceae
• Unique in brain abscess formation

5

• Meningitis
• inflammation of the meninges the thin,
  membranous covering of the brain and spinal cord
• Blood-brain barrier (BBB)
• an arrangement of cells w/in the brain blood
  vessels preventing the passage of toxic
  substances from the blood into the brain
  penetration by bacteria necessary for CNS
  infections

6
Materials/Methods

• Brain capillaries isolated and homogenized to
  microvessels
• Microvessels purified and examined for viability
• Human endothelial cells cultured
• Human brain microvascular endothelial cell
  (HBMEC) transfection with pBR322 based plasmid
  containing simian virus large T protein

7

• Characterization of brain endothelial cell
  monolayers
• -Morphology
• -Factor VIII-Rag
• -Acetylated low density lipoprotein (AcLDL)
  uptake
• -gamma glutamyl transpeptidase
  (GGTP)

8
Rationale

• To characterize primary and transfected (T)HBMEC
  morphologically and functionally to determine if
  the THBMEC serves as a reliable model of the BBB

9
Transfected human brain microvascular endothelial
cells (THBMEC)

• Morphology of THBMEC DiI-AcLDL uptake

Figure 1 on Handout
10
THBMEC fixed and stained for specific proteins
characteristic of primary HBMEC

• Factor VIII-Rag GGTP
  SV40 large T antigen

Figure 2 on handout
11
THBMEC may be useful for studying CNS infection
pathogenesis

• THBMEC
• Stained positive for Factor VIII-Rag
• Took up DiI-AcLDL
• Stained strongly positive for gamma glutamyl
  transpeptidase (GGTP)
• Presence of large T in transfected cells gt95

12
Materials/Methods

• Bacterial invasion in (T)HBMEC
• E. coli K1 strains C5 and E44
• Citrobacter freundii
• 107 bacteria added
• Gentamicin treatment
• Non-invasive E. coli strain (HB101) used as
  control
• Percent invasion 100 x ( bacteria
  recovered)/( bacteria inoculated)

13
Rationale

• To determine the interaction between
  meningitis-causing bacteria, E. coli K1 and C.
  freundii, and the primary and transfected HBMEC

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Invasion of E. coli K1 strains into primary and
transfected HBMEC
Incub. for 8 hrs.
Invasion ( inoculum)
E44
Bacteria added
15
Invasion of bacteria into primary and transfected
HBMEC C. freundii vs. E. coli HB101
Invasion at 2h
Invasion ( inoculum)
Bacteria added
16
Data Recap

• Invasion of HBMEC significantly higher with E.
  coli strains E44 and C5
• No significant difference of E. coli strains of
  HBMEC and THBMEC
• C. freundii invasion greater than HB101
• C. freundii invasion similar in primary and
  transfected HBMEC

17

• Transfected HBMEC exhibit numerous similarities
  with primary HBMEC.
• Further evidence that THBMEC serve as a reliable
  BBB model

18
Materials/Methods

• Transcytosis of bacteria across HBMEC monolayers
• Bacteria added to apical (T)HBMEC
• HBMEC grown on Transwell filters
• Bottom compartment monitored
• Transcytosis measured 8 hrs. after bacteria
  addition

19
Rationale

• To compare THBMEC and primary HBMEC transcytosis
  of E. coli K1and C. freundii

20
Transcytosis of E. coli across monolayers of
(T)HBMEC
determined at 8 hrs.
Transcytosis ( inoculum)
E44
Bacteria added
21
Data Recap

• E. coli K1 strains transversal significantly
  higher than HB101
• E. coli K1 HBMEC transversal do not differ
  significantly from THBMEC

22
Transcytosis of C. freundii across monolayers of
(T)HBMEC
determined at 2 hrs.
Transcytosis ( inoculum)
Bacteria added
23
Data Recap

• C. freundii effective in transversal of both
  HBMEC and THBMEC
• 18 inoculum found in lower compartment
• Transversal properties of E. coli K1 and C.
  freundii relative to HB101 maintained

24
Conclusions

• THBMEC shares many similar characteristics as
  primary HBMEC
• Morphology
• GGTP
• Acetylated low-density protein (AcLDL) uptake
• Factor VIII-Rag

25

• CSF isolates of E. coli K1 strains E44 and C5 are
  able to similarly invade both primary and
  transfected HBMEC
• Both strains were able to transcytose both
  monolayers
• C. freundii possesses ability to invade and
  transcytose both THBMEC and HBMEC

26

• Transfected HBMEC by large T protein showed no
  significant characteristical differences compared
  to primary HBMEC

• Can therefore act as a reliable BBB model
• to continue studying unclear pathogenesis of
• CNS infections

27
References

• Ades EW, Candal FJ, Swerlick RA. 1992. HMEC-1
  establishment of an immortalized human
  microvascular endothelial cell line. Journal of
  Investigative Dermatology. 99683-690.
• Badger JL, Stins MF, Kim KS. 1999. Citrobacter
  freundii invades and replicates in human brain
  microvascular endothelial cells. Infection and
  Immunity. 674208-4215.
• Stins MF, Badger J, Kim KS. 2001. Bacterial
  invasion and transcytosis in transfected human
  brain microvascular endothelial cells. Microbial
  Pathogenesis. 3019-28.