Involvement of membrane Lipid Rafts in the internalization of Peudomonas aeruginosa by immortalized corneal epithelial cells (hTCEpi)

Abstract

Abstract: : Purpose: Recently the internalization of P. aeruginosa (PA) in nasal and tracheal epithelial cells (in vivo, in vitro) has been shown to involve the formation of sphingolipid–rich plasma membrane domains forming lipid raft platforms (Nat. Med. 9:322, 2003). The purpose of this study was to evaluate the role of lipid raft formation in the internalization of PA in human corneal epithelium. Methods: Human corneal epithelial cells immortalized with human telomerase reverse transcriptase (hTCEpi) were cultured in KGM–2 medium for 2days. Cells were then incubated with the cholesterol metabolism inhibitor Methyl–ß–cyclodextrin (MßCD, 0–10 mM) prior to the incubation with PA. After rinsing with basal medium, the cells were incubated with PA (5x10⁷CFU/mL) for various times to allow the bacterial internalization. Bacterial internalization was assayed by the treatment with gentamicin (200µg/mL) followed by cell disruption and bacterial culture on Mueller Hinton agar plates. Colony forming units (CFU) were counted 20 hours after plating. Lipid raft formation was assessed by staining with the FITC–conjugated ß–subunit of cholera toxin (ß–CT), followed by confocal microscopy. Results: In contorol cultures, ß–CT, which binds to the lipid raft component ganglioside GM1, showed diffusely weak staining of the hTCEpi plasma membrane. After exposure to PA, ß–CT intensely stained focal regions of the plasma membrane that associated with clusters of PA. Focal staining by ß–CT appeared to increase in number and frequency over time from 15min to 3 hours. Increasing of ß–CT staining over time was associated with the increased bacterial internalization from 1.7±0.1x10³ CFU at 30min to 2.8±0.4x10⁵ CFU at 3hours. Addition of MßCD significantly blocked the internalization in a dose dependent manner (p<0.01). Conclusions: These results show P. aeruginosa are internalized in hTCEpi cells through the formation of lipid rafts. These findings also suggest that hTCEpi cells may be used as a model for studying the molecular mechanisms of Peudomonas infection in humans.

Keywords: cornea: epithelium • contact lens • Pseudomonas