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 Citrobacter: 25-50% (neonatal meningitis) • Excellent models: Bacterial penetration of the blood brain barrier (BBB)--- E. coli K1 and group B streptococcus (GBS) [low brain abscess formation] • Citrobacter [high brain abscess formation]: indicative of a different pathogenic mechanism

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 To obtain the bacteria and HMBEC: • Bacterial Strains were spontaneous rifampicin resistant mutants • Derived from urinary tract isolates • Grown aerobically in brain heart infusion broth. • HMBEC cultures isolated from a brain biopsy of adult female with epilepsy • Cultures took up fluorescently labeled low-density lipoprotein

7. To determine the ability of C. freundii to invade HBMEC: • 107 bacteria added • Intracellular bacteria determined 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. Transmission electron microscopy (TEM) to characterize the interaction btw C. freundii and HBMEC including replication ability • HBMEC incubated w/ C. freundii for various times • HBMEC monolayer washed at specific times 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, 45 min • B) Intracellular (vacuole), 1.5 hour • C)Replication, 4 hours

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. Effects of various eukaryotic inhibitors on C. freundii invasion 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 • Microfilament inhibitor cytochalasin D • Compared HMBEC treated and untreated with cytochalasin D inhibitor • Cytochalasin D 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 • Microtubule inhibitors used to show invasion effects of HBMEC • Nocodazole • Vincristine

19. Role of HBMEC protein synthesis • To examine whether de novo eukaryotic protein synthesis plays a role in C. freundii invasion • Protein synthesis inhibitor, cyclohexamide

20. Endosome acidification • To examine the role of endosome adicification in the C. freundii invasion process • Endosome acidification inhibitor, monensin • Monensin - cationic ionophore known 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) Incubated for 15 min C) Incubated for 30 min D) Nocadazole-pretreated (30 min) E) Cytochalasin D-pretreated

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

27. Conclusions • Invasion assay results 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 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 treatments • 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