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. 30:19-28. 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 >95%

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

14. 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. 99:683-690. • Badger JL, Stins MF, Kim KS. 1999. Citrobacter freundii invades and replicates in human brain microvascular endothelial cells. Infection and Immunity. 67:4208-4215. • Stins MF, Badger J, Kim KS. 2001. Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells. Microbial Pathogenesis. 30:19-28.