Citrobacter freundii invades and replicates in human brain microvascular endothelial cells

Abstract

Neonatal bacterial meningitis remains a disease with unacceptable rates of morbidity and mortality despite the availability of effective antimicrobial therapy. Citrobacter spp. cause neonatal meningitis but are unique in their frequent association with brain abscess formation. The pathogenesis of Citrobacter spp. causing meningitis and brain abscess is not well characterized; however, as with other meningitis-causing bacteria (e.g., Escherichia coli K1 and group B streptococci), penetration of the blood-brain barrier must occur. In an effort to understand the pathogenesis of Citrobacter spp. causing meningitis, we have used the in vitro blood-brain barrier model of human brain microvascular endothelial cells (HBMEC) to study the interaction between C. freundii and HBMEC. In this study, we show that C. freundii is capable of invading and trancytosing HBMEC in vitro. Invasion of HBMEC by C. freundii was determined to be dependent on microfilaments, microtubules, endosome acidification, and de novo protein synthesis. Immunofluorescence microscopy studies revealed that microtubules aggregated after HBMEC came in contact with C. freundii; furthermore, the microtubule aggregation was time dependent and seen with C. freundii but not with noninvasive E. coli HB101 and meningitic E. coli K1. Also in contrast to other meningitis-causing bacteria, C. freundii is able to replicate within HBMEC. This is the first demonstration of a meningitis-causing bacterium capable of intracellular replication within BMEC. The important determinants of the pathogenesis of C. freundii causing meningitis and brain abscess may relate to invasion of and intracellular replication in HBMEC.

FIG. 1

TEM demonstrating C. freundii invasion of HBMEC at a magnification of ×14,000. (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.

FIG. 2

Effects of different eukaryotic cell function inhibitors on C. freundii invasion of HBMEC. The inhibitors at indicated concentrations were added before addition of the bacteria and were present until gentamicin treatment (see Materials and Methods). Results are presented as relative invasiveness (see Materials and Methods) and represent the mean ± standard deviation of at least three individual experiments performed in duplicate.

FIG. 3

Confocal immunofluorescence microscopy of gfp-expressing C. freundii and rhodamine-stained HBMEC microtubules. The superimposed images were generated by the LSM conversion program and subsequently labeled in Adobe Photoshop. (A) No bacteria added to HBMEC; (B to E) C. freundii incubated for 15 min with HBMEC (B), for 30 min with HBMEC (C), for 30 min with nocadazole (5 μg/ml)-pretreated HBMEC (D), and for 30 min with cytochalasin D (0.25 μg/ml)-pretreated HBMEC (E). HBMEC microtubules were stained by indirect immunofluorescence as described in Materials and Methods. All panels are of equal magnification (×4,000).

FIG. 4

Transcytosis of C. freundii across polarized HBMEC monolayers. HBMEC were grown to confluence on Transwell filters as described in Materials and Methods. Bacteria were added to the apical side. Samples were collected from the basolateral chambers at indicated times and plated for CFU (right axis). Simultaneously, passive diffusion was measured via ³H-inulin (left axis). ⧫, no bacteria; ■, C. freundii; ▴, HB101. Results are presented as a representative assay of four independent experiments, each performed in triplicate and all yielding similar results.