Citrobacter freundii Invades and Replicates in Human Brain Microvascular Endothelial Cells

1. Citrobacter freundii Invades and Replicates in Human Brain Microvascular Endothelial Cells Badger J, Stins MF, and Kim KS. 1999. Infection and Immunity. 67:4208-4215. Presented by Jess Jung

2. General Rationale of Experiment • Morbidity and mortality rate of Citrobacter causing meningitis is extremely high (25-50% fatality rate associated with neonatal meningitis) • Previously studied E. coli K1 and group B streptococcus (GBS) are the two leading causes of neonatal bacterial meningitis, excellent models for studying bacterial penetration of the blood brain barrier (BBB) • Unlike Citrobacter, they have low association of brain abscess formation, indicative that Citrobacter uses a different pathogenic mechanism for crossing the BBB

3. Purpose • To better understand the potential interactions of Citrobacter with human brain microvascular endothelial cells (HBMEC), the model used to represent the human blood brain barrier

4. Terms to Know • Citrobacter freundii (C. freundii) – member of the family Enterobacteriaceae, gram-negative bacilli • Meningitis – inflammation of the meninges; the thin, membranous covering of the brain and spinal cord • Brain Abscess – focal, intracerebral infection that begins as a localized area of cerebritis, developing into a collection of pus surrounded by well-vascularized capsule

5. 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 • Human Brain Microvascular Endothelial Cells (HBMEC) – isolated from a brain biopsy, used as the BBB model to study the interaction between C. freundii and HBMEC

6. Materials and Methods First procedure: to obtain the bacteria and HMBEC • Bacterial Strains were spontaneous rifampicin resistant mutants derived from urinary tract isolates. Bacteria grown aerobically for 14h at 37C in brain heart infusion broth. • HMBEC cultures were isolated from a brain biopsy of an adult female with epilepsy. Cultures took up fluorescently labeled low-density lipoprotein and demonstrated their brain endothelial cell properties.

7. Second procedure: to determine the ability of C. freundii to invade HBMEC • 107 bacteria added to a well with monolayer of HBMEC at a multiplicity of infection of 100. • Intracellular bacteria determined after the extracellular bacteria eliminated by incubation of the monolayer with experimental medium with gentamicin • Noninvasive E. Coli used as a negative control • Percent invasion= 100 x [(# bacteria recovered)/(# bacteria inoculated)]

8. % invasion Time (hours) btw addition of gentamicin and enumeration of intracellular bacteria % Invasion of C. freundii inHBMEC

9. Recap of data • C. freundii does, in fact, penetrate the human brain microvascular endothelial cells (HBMEC) used as a model of the BBB in this experiment.

10. Fourth procedure: using transmission electron microscopy (TEM) to characterize the interaction btw C. freundii and HBMEC including replication ability • HBMEC incubated w/ C. freundii for various times • At certain times, HBMEC monolayer washed four times and gently scraped from surface • Cell flurry centrifuged • Pellet resuspended and fixed

11. Cells washed, postfixed, rinsed, dehydrated through ethanol solutions, and embedded in polypropylene oxide • Ultrathin sections cut out, mounted on grids, and stained to be examined via TEM

12. TEM showing C. freundii invasion of HBMEC at x14,000 magnification • A) Extracellular C. freundii attached to HBMEC • B) Intracellular C. freundii found within membrane-bound vacuole-like structures • C) C. freundii replicating within vacuole-like structures

13. Data Recap • Entry of C. freundii into HBMEC after a 45 min incubation period • C. freundii found intracellularly (in vacuole-like structures of the HBMEC) • Vacuole-like structures of HBMEC contain multiple C. freundii, indicative of replication

14. Fifth procedure: analysis of the effects of various eukaryotic inhibitors on C. freundii invasion of the HBMEC to identify the cellular components necessary for invasion • Examined the roles of: • Microfilaments • Microtubules • HBMEC protein synthesis • Endosome acidification

15. Microfilaments • Cytoskeletal structure that enables the cell cytoplasm to move • Composed of a polymer of actin that can rapidly assemble and disassemble, causing motion

16. Role of microfilaments in HBMEC invasion • Various concentrations of inhibitor cytochalasin D used to pretreat HBMEC • Compared abilities of C. freundii to invade treated and untreated HBMEC • Cytochalasin D used because it causes depolymerization in eukaryotic cells

17. Microtubules • Long, hollow cylinders composed of protein subunits called tubulin • Thickest of cytoskeletal structures • Form mitotic spindles, the machinery that partitions chromosomes between two cells in the process of cell division

18. Role of microtubules in HBMEC invasion • To establish involvement of microtubules in C. freundii invasion of HBMEC, invasion assays were performed with different microtubule inhibitors • Pretreatment of HBMEC with nocodazole, a microtubule depolymerizing agent, and vincristine, a microtubule stabilizing agent, used to show invasion effects of HBMEC

19. Role of HBMEC protein synthesis • To examine whether de novo eukaryotic protein synthesis plays a role in C. freundii invasion, assays were done with cycloheximide treated HBMEC and compared to untreated HBMEC

20. Endosome acidification • To examine the role of endosome adicification in the C. freundii invasion process, the inhibitor, monensin was used in invasion assays • Monensin is a cationic ionophore that has been shown to increase the pH of intracellular vacuoles

21. The following data depicts the effect of various cell function inhibitors including: • Microfilaments • Microtubules • Protein synthesis • Endosome acidification

22. C. freundii invasion of HBMEC effected by various cell function inhibitors

23. Data Recap • C. freundii invasion is dependent on microfilaments, microtubules, protein synthesis, and endosome acidification

24. Microtubule aggregation associated with C. freundii invasion • To further confirm the apparent role of microtubules in C. freundii invasion of HBMEC, fluorescence microscopy was used to examine whether there were changes in the tubule networking

25. Confocal immunofluorescence microscopy of gfp-expressing C. freundii and rhodamine- stained HBMEC microtubules A) no bacteria added to HBMEC B) C. freundii incubated for 15 min w/ HBMEC C) C. freundii incubated for 30 min w/ HBMEC D) Incubated for 30 min w/ nocadazole-pretreated HBMEC E) Incubated for 30 min w/ cytochalasin D-pretreated HBMEC

26. Major indication of data • Microtubules aggregate after they come in contact with C. freundii

27. Conclusions • Results from invasion assays performed in the presence of various eukaryotic cellular inhibitors suggest that C. freundii invasion into HBMEC is dependent on microfilaments, microtubules, de novo protein synthesis and endosome acidification • C. freundii can survive and replicate intracellularly in vitro

28. TEM analysis revealed the intracellular location of individual and multiple C. freundii cells within single membrane vacuole-like structures • Microtubule inhibitors (both deploymerizing and stabilizing agents) significantly decreased the ability of HBMEC to take up C. freundii • Confocal immunofluorescence microscopy showed that microtubules aggregate after HBMEC come in contact with C. freundii

29. Relevance • Understanding the pathogenesis of Citrobacter causing meningitis and brain abscess will be helpful in determining remedies • Also, pathogenesis may relate to invasion and intracellular replication in HBMEC

30. References Nester EW, Anderson DG, Roberts CE, Pearsall NN, Nester MT. Microbiology: A Human Perspective, 3rd edition. Copywrite 2001 by the McGraw-Hill Companies, pp 77. Gale Encyclopedia of Medicine. 1996. pp 1900. Badger, J. L., and K. S. Kim. 1998. Environmental growth conditions influence the ability of Escherichia coli K1 to invade brain microvascular endothelial cells and confer serum resistance. Infect. Immun. 66:5692-5697 Kim, K. S., H. Itabashi, P. Gemski, J. Sadoff, R. L. Warren, and A. S. Cross. 1992. The K1 capsule is the critical determinant in the development of Escherichia coli meningitis in the rat. J. Clin. I nvestig. 90:897-905